In the summer of 1995, after befriending fellow Universal Studios stage performer, Chris Kirkpatrick, who was singing in a doo-wop group, he became the fourth member of NSYNC, along with Justin Timberlake and JC Chasez. Then Lance Bass was added to the group that would become NSYNC. In 2001, Fatone co-starred with *NSYNC bandmate Lance Bass in the Miramax film On the Line, he guest starred with NSYNC on a 2001 episode of The Simpsons episode New Kids on the Blecch and also did voice work with Bass on the same episodes of Disney's Kim Possible and Adult Swim's Robot Chicken.

Fatone co-starred in the 2002 dramatic comedy motion picture My Big Fat Greek Wedding as Toula's cousin Angelo, appeared in the show My Big Fat Greek Life, and reprised the role again in the 2016 sequel My Big Fat Greek Wedding 2. He acted in the critically acclaimed movie The Cooler. He has had minor roles in both the experimental film Red Riding Hood and Homie Spumoni, the latter alongside Jamie-Lynn Sigler. Fatone has also been on Broadway performing leads in Rent and in a revival of Little Shop Of Horrors.

On September 9, 2004, Fatone married his on-and-off girlfriend of 10 years, Kelly Baldwin. The ceremony took place in Oheka Castle on Long Island, New York and was attended by all of Fatone's bandmates. Fatone and his wife have two daughters, Briahna born March 21, 2001 and Kloey Alexandria born January 11, 2010. Fatone's best friend, Lance Bass, is their godfather.

On February 1, 2007, the ABC television network announced that Fatone would participate in season 4 of the American version of Dancing with the Stars, which debuted March 19, 2007. He ended up placing second, losing the mirror ball to Apolo Anton Ohno and Julianne Hough. He competed with professional partner Kym Johnson. Fatone participated in the 15th season of Dancing with the Stars for a second chance to win a mirror ball trophy, again dancing with Kym Johnson. They were the second couple eliminated.

On October 18, 2009, Fatone attended the TNA Wrestling's biggest annual pay-per-view event TNA Bound for Glory and co-hosted the live pre-show alongside Jeremy Borash. This was not the first time Fatone has been linked to TNA as he has been spotted at Universal Orlando where TNA tapes their weekly television program.

In the twelfth season of Family Feud in 2010, Fatone became the announcer with Steve Harvey as the host for that season.

In 2011, he started hosting the Live Well Network series My Family Recipe Rocks. In the same year he was in a horror film called "Inkubus."

In January 2012, he was one of eight celebrities participating in the Food Network reality series Rachael vs. Guy: Celebrity Cook-Off. In 6 weeks he was eliminated, but he won $5000 for his charity, the Fatone Family Foundation. He also hosted the Las Vegas show The Price Is Right at Bally's Las Vegas, as a Celebrity guest host.

On August 25, 2013, the members of NSYNC regrouped for a one-off performance at the 2013 MTV Video Music Awards. They performed a medley of "Girlfriend" and "Bye Bye Bye". After the performance, Lance Bass said that the group does not have plans at the moment for a reunion tour or new music.

In August 2014 Fatone hosted the family reality series, Parents Just Don't Understand, seen on the Hub Network, which was later re-branded as Discovery Family.

In 2015, Fatone made a couple of appearances as himself in The Jack and Triumph Show.

In November 2016, Fatone made another appearance on Impractical Jokers for the Nitro Circus Spectacular episode.