Seven-year-old Tim Templeton (voice of Miles Bakshi) has an active imagination. He has all the love in the world from his parents Ted and Janice (voices of Jimmy Kimmel and Lisa Kudrow), who play with him in all of his adventures. Ted and Janice work at PuppyCo in the marketing department, which Tim thinks is a cool job. As Tim's adult self (voice of Tobey Maguire) narrates, things became different when Tim's parents asked him how he would feel about having a little brother. Young Tim tells them he is enough, unaware that his mother is pregnant. Within the foundations of BabyCorp, numerous babies are being born (never quite explained how) and are being processed through a conveyor belt. One small baby in particular goes through the motions, getting a fresh diaper and a pacifier. The tickle machine determines where babies are placed.
If they laugh, they are sent to families. If they don't react, they are sent to management. The baby boy is pushed into management and is given his own suit, watch, and briefcase before being assigned to his cubicle. Tim anxiously looks out the window to see his parents returning in a taxi with their new baby. It's the same baby from before, The Boss Baby (voice of Alec Baldwin), strutting up to the front door with his briefcase, to Tim's bewilderment.
From the moment he enters the house, Boss Baby begins to command the household. Ted and Janice are so busy trying to please Boss Baby that Tim starts to feel shut out. However, Tim seems to be the only one that finds something suspicious about Boss Baby, with his suit and briefcase standing out. Tim sneaks into Boss Baby's room that night and hears him speaking with the voice of a grown man on the phone.
He appears to be conspiring with someone until Tim catches him and forces Boss Baby to admit that he can talk. Although Tim thinks he's got something on him, Boss Baby is unfazed and pulls out a file on Tim, stating he knows things like how Tim's middle name is Leslie. Boss Baby warns Tim that if he doesn't shape up, his parents can "fire" him. Tim sets out to prove that Boss Baby is up to no good. He spies on Boss Baby as he has a playdate with other baby associates - Jimbo (voice of David Soren), Staci (voice of ViviAnn Yee), and the Triplets (voice of Eric Bell, Jr. ). Boss Baby gives a presentation about BabyCorp's enemy, PuppyCo. The CEO, Francis Francis (voice of Steve Buscemi), is set to unveil their newest puppy, which could spell trouble for babies everywhere.
The other babies start to get anxious when Boss Baby starts to ask what they learned from the presentation. They put on their baby act for the parents when they come around. Tim has a tape of the babies' conversation, but the babies catch him and set off to get the tape. This leads to Tim having to evade the babies in the backyard as he tries to show the tape to his parents. Boss Baby rides a small car to get Tim while the other babies go after him. Tim traps Staci in the sandbox while Jimbo rides through a fence, and the Triplets are also stopped. Tim runs back into the house, just as the adults are walking outside to see their kids in a chaotic scene. Tim runs to his parents' office and finds Boss Baby sitting in their chair.
He demands the tape or else he will harm Tim's favorite stuffed doll, Lamb-Lamb. They fight for it, and they accidentally rip Lamb-Lamb's head off. Tim gets angry and grabs Boss Baby, putting him in his walker and threatening to launch him out the window. Ted and Janice walk in and see this, causing Tim to launch the tape into the street instead. Since they think he's trying to harm his brother, Tim's parents ground him. Boss Baby goes into Tim's room as he is crying. He decides to finally explain his situation to the boy.
Boss Baby gives Tim one of his pacifiers and tells him to suck on it until he finds the answers he's looking for. The two are then teleported to BabyCorp as projections, watching how everything works. Boss Baby explains how he got into management and how there have been many other great Boss Babies before him. His own boss, Big Boss Baby (voice of Edie Mirman), runs her department with an iron fist. Boss Baby aspires to be like the most famous baby, Super Colossal Big Fat Boss Baby, so he can get the dream promotion with his own office and personal potty. He also explains how the babies stay babies, which is by drinking a special formula that stops them from aging. When Big Boss Baby is seen demanding results from Boss Baby, he panics and snaps himself and Tim back to reality. He explains that if he doesn't succeed in his mission to bring down PuppyCo, he will get fired and be forced to live with the Templetons forever.
Tim then agrees to help Boss Baby, but only so he can leave. The boys then try to pretend for their parents that they love each other and are playing together, while also working together to find info on the new puppy. Ted and Janice later invite the boys to "Bring Your Kid To Work Day" at PuppyCo. Once there, Ted and Janice invite Tim to spend time with them while they leave Boss Baby in the puppy pit.
Tim thinks about it but decides to stay with Boss Baby so they can look for the new puppy. Tim has Boss Baby dress up as a puppy to get past security. They go into a room and find a file on the new puppy, but they must replace it with another file. It works until Boss Baby gets drool on the replacement, setting off a trap. The puppy file then turns out to be a fake. The boys are caught by Eugene (voice of Conrad Vernon), a man dressed as PuppyCo's mascot.
Eugene brings the boys to meet Francis. He reveals to them that he is Super Colossal Big Fat Boss Baby, now all grown up. As a baby, he got the promotion he wanted and was at the top of his game. But because he is lactose intolerant, the formula stopped working for him, and he was booted out of BabyCorp. Now, he plots revenge against them by creating the Forever Puppy, which will stay a cute puppy for all time, stealing all the love away from babies. The only thing Francis needs is some formula for the puppy to be created, which Eugene takes out of Tim's backpack. He then sends the boys away. Ted and Janice leave the boys with Eugene (disguised as a housekeeper) while they go with Francis to Las Vegas to unveil the Forever Puppy.
Without the formula, Boss Baby starts acting like a real baby. Tim and Boss Baby come up with a plan to escape Eugene by faking Boss Baby being sick and spewing fake vomit all over Eugene. The boys then ride away on Tim's bike, while Eugene chases them on a wagon. Boss Baby calls Staci and has her round up Jimbo and the Triplets to help the boys. Together, they get Tim and Boss Baby to the airport while stopping Eugene. Unfortunately, the plane for Vegas takes off before the boys can stop their parents. They then argue with both throwing the blame on one another, with Tim blaming Boss Baby for being a normal baby at the worst moment, while Boss Baby argues that they would have gotten there faster if Tim could ride a bike without training wheels. Tim tells him he knows nothing about having a real family, leading to Boss Baby ditching Tim and leaving him alone at the airport.
Hours later, Boss Baby calls Tim on a phone right next to him. They reconcile and resolve to work together to get to Vegas themselves since they now know they need each other. The boys follow Elvis impersonators for a convention to Vegas, unaware that Eugene is following them as well. On the plane ride to Vegas, Tim pretends that he and Boss Baby are the captain's kids to get special perks. He shows Boss Baby how to have fun by engaging in a playtime scenario where they are pirates hijacking a ship.
Boss Baby really gets into it and has actual fun for the first time in his life. The boys make it to Vegas and arrive at the convention center for the unveiling. Francis reveals the Forever Puppy to the public with Ted and Janice helping him. When the boys reveal themselves, Francis locks Ted and Janice inside a giant heart and wheels them away. Eugene tries to get the boys as they run after Francis, who is set to literally launch the Forever Puppies with a rocket.
Eugene runs through a set of tubes to get to the boys, but they outsmart him and cause him to fall into a giant litter box to get scratched up by cats. The boys catch up to Francis, who tries to throw them over the ledge. Tim and Boss Baby re-enact their pirate game and throw Francis into a vat of formula. They rush to save their parents and get the puppies out of the rocket. Boss Baby then becomes a normal baby and is stuck up on the rocket until Tim sings Paul McCartney's "Blackbird" too get the baby down. Tim then protects his brother as the empty rocket launches.
Tim runs to the formula vat and gives Boss Baby some of it to bring him back to normal. Francis then emerges from the vat, now reverted back to Super Colossal Big Fat Boss Baby. He threatens the boys until Eugene picks him up and gives him a pacifier. Eugene tells the boys that he will raise Francis the right way this time. The boys go home, and Boss Baby is able to leave to get his promotion. The other babies from BabyCorp wipe Ted and Janice's memories of the baby and remove any evidence that he was there, but Tim chooses not to forget him. Boss Baby returns to work as a hero and he gets his promotion like he wanted. However, both boys miss one another and don't feel happy even though they got what they wanted.
Tim writes a letter to Boss Baby and sends him some of his toys. Boss Baby gets excited and he runs into the main room to play with the computer and basically quit. He goes through the conveyor belt and has himself sent down the "family" chute. Sometime later, Boss Baby returns to the family as a normal baby, now named Theodore Lindsay Templeton. Tim is excited to see him, as the baby appears to remember him. He runs up and grabs his new baby brother. The two then go on to have many adventures.
We jump to the future with Adult Tim having been telling the whole story to his daughter, who is about to meet her own baby sister. With them is Adult Ted, still having a nice brotherly bond with Tim. Tim's daughter looks in the delivery room and sees her baby sister. To her surprise, the baby rises from her bed and is wearing a suit.