Tim Templeton is now fully grown and lives with his wife Carol and their two daughters, 7-year-old Tabitha and infant Tina. Tim's younger brother Ted is now a successful CEO and is never around. Tabitha is exhibiting more grown-up behavior, and one night while Tim wonders about the person his daughter is becoming, he hears something from Tina's room. He discovers that Tina is from BabyCorp, just as Ted once was, and that she has been assigned to get Ted there for a special mission. Tim refuses to call Ted, saying that he will never come, and encourages Tina to go back to sleep. However, Tina leaves a fake voicemail for Ted, luring him to the Templeton's house.
The next morning, Ted arrives and Tim explains to him that Tina is a BabyCorp dispatch. Tina introduces the brothers to a new formula that will allow them to turn back into children for 48 hours in order to infiltrate Tabitha's school and figure out what Dr. Erwin Armstrong, founder and principal of the school, is planning behind parents' back.
At the school, Tim, now as his 7-year-old self, follows Tabitha to her class while baby Ted is placed with other babies. Ted rallies the babies to help him get out of the playroom so that he can go to Armstrong's office to investigate. Tim tries to get sent to the principal's office by disrupting class, but is instead put in "The Box" for timeout. Ted discovers that Armstrong is actually a baby himself, having run away from home after realizing that he was smarter than his parents and now makes money by creating popular phone apps. His ultimate plan is to get rid of every parent on B-Day, so that they cannot tell their children what to do anymore.
On the night of the holiday pageant, where Tabitha is supposed to sing a solo, the brothers and Tina plan to expose Armstrong. However, they learn that B-Day is set to happen that night through Armstrong's new app, QT-Snap, which will hypnotize the parents into mindless zombies. Both Tim and Ted are caught by Armstrong's ninja babies and are put in The Box, which slowly starts to fill with water. Tabitha sings her solo, but when she sees that Tim has not showed up, she runs off the stage crying. She is consoled by Tina, who reveals her identity and her mission. Tabitha agrees to help her younger sister by getting to the server and shutting down QT-Snap before it can go worldwide. Ted is able to call Precious, Tabitha's pet pony, into the school, as she breaks them out of The Box.
Tim and Ted reach the server first, but they are stopped by Armstrong, who calls the zombie parents for backup. While the brothers hold them back as the formula starts to wear off, Tina and Tabitha get up to the server. Tabitha is able to hack in and pull up the shutdown screen, but is interrupted by Armstrong. The sisters then set off a candy lava volcano using Mentos and soda, destroying the servers and turning all parents back to normal. Tina then reveals that bringing Tim and Ted back together was her true mission. The whole Templeton family gathers to celebrate Christmas, while Armstrong returns to his own family.
Alec Baldwin as Ted Templeton Jr. / Boss Baby, a former executive of BabyCorp, Tim's younger brother, Tina and Tabitha's uncle, Carol's brother-in-law, and Ted Sr. and Janice's younger son.
James Marsden as Tim Templeton, Ted Jr.'s older brother, Carol's husband, Tina and Tabitha's father, and Ted Sr. and Janice's older son. Marsden replaces Tobey Maguire and Miles Bakshi in the first film, where Maguire voiced the adult Tim and Bakshi voiced the younger Tim.
Amy Sedaris as Tina Templeton, an undercover and new executive of BabyCorp, Tim and Carol's younger daughter, Tabitha's younger sister, Ted's younger niece, and Ted Sr. and Janice's younger granddaughter.
Ariana Greenblatt as Tabitha Templeton, Tina's older sister, Tim and Carol's highly intelligent older daughter, Ted's older niece, and Ted Sr. and Janice's older granddaughter. Greenblatt replaces Nina Zoe Bakshi from the first film.
Jeff Goldblum as Dr. Erwin Armstrong, an intelligent baby with a plot to remove all grownups.
Eva Longoria as Carol Templeton, Tim's wife, Ted Jr.'s sister-in-law, Ted Sr. and Janice's daughter-in-law, and Tina and Tabitha's mother.
Jimmy Kimmel as Ted Templeton Sr., Janice's husband, Ted Jr. and Tim's father, Carol's father-in-law, and Tina and Tabitha's grandfather.
Lisa Kudrow as Janice Templeton, Ted Sr.'s wife, Ted Jr. and Tim's mother, Carol's mother-in-law, and Tina and Tabitha's grandmother.
James McGrath as Wizzie, Tim's Gandalf-esque alarm clock from his childhood.
American computer-animated comedy film loosely based on the 2010 picture book The Boss Baby and its 2016 sequel The Bossier Baby by Marla Frazee