In 2006, Urie began appearing in the ABC dramedy Ugly Betty as Marc St. James, the assistant of Wilhelmina Slater, played by Vanessa Williams.

[10] Urie has returned often to his theater roots, including directing a one-night celebrity-performed staging of Howard Ashman's unproduced musical Dreamstuff.

The musical was reimagined by Howard's partners Marsha Malamet and Dennis Green and performed at Los Angeles's Hayworth Theatre as part of the Bruno Kirby celebrity reading series.

[citation needed] He has also been on Live with Regis and Kelly and has also starred in the 2008 Disney blockbuster production Beverly Hills Chihuahua as the voice of Sebastian.

Urie originated the role of Rudi Gernreich in the 2009 off-Broadway play The Temperamentals, about the foundation of the early LGBT rights organization the Mattachine Society.

[11] In January 2012, Urie made his Broadway debut, joining the cast of the second revival of How to Succeed in Business Without Really Trying in the role of Bud Frump.

[13] In 2012, Urie also starred as the mysterious limo driver James in the film adaptation of Wendy Mass's children's book Jeremy Fink and the Meaning of Life, written and directed by Tamar Halpern.

The multi-camera comedy, from Will & Grace creators Max Mutchnick and David Kohan, centered on lifelong friends and business partners – one straight and one gay.

[19][20] He had a recurring role as Redmond, the gossipy book agent, in the popular TVLand drama-comedy series Younger, produced by Darren Star.

[23] On September 13, 2019, it was announced that Urie would once again team up with his Ugly Betty co-star Becki Newton on a sitcom project for CBS and Warner Bros. Television called Fun, in which he would both co-star and serve as a co-executive producer with creator Michael Patrick King and fellow Ugly Betty showrunners Tracy Poust and Jon Kinnally.