Pennette's career began on the sitcom Kate & Allie from where he moved on to other projects including Dave's World and Dear John. He went on to create and write for many of his own series: Caroline in the City, Union Square, Conrad Bloom and All About the Andersons. Most notably, though, were the short-lived medical drama Inconceivable co-created with Oliver Goldstick, inspired by both of their surrogate pregnancy experiences,and the short-lived midseason sitcom Crumbs, an autobiographical portrayal of his family experiences and closeted young adulthood. His other writing and producing work from 2003 onwards includes serving as executive producer and co-executive producer on the television series I'm with Her and the sitcom What I Like About You, for both of which he also wrote a number of episodes. He also wrote the script for and an the executive producer of the 2007 Football Wives pilot, which was originally intended to be a television series but was later declined by the American Broadcasting Company in favour of seven other pilots with lower budgets, as the ABC claimed that the pitched budget for Football Wives was too high for a midseason pick-up. The series was set to be a U.S. version of the popular British soap opera Footballers' Wives.

He was a member of the writing panel and an executive producer/co-showrunner on the dramedy series Ugly Betty, initially having been a co-executive producer. Ugly Betty's creator and other showrunner Silvio Horta was named AfterElton.com's Man of the Year for 2007 for the show's positive portrayal of LGBT issues—including a possibly gay teenager, a transgender man-turned-woman and a gay male assistant with a homophobic mother—and had to say about Pennette, also gay: "Marco Pennette, my co-showrunner and Executive Producer, deserves a special thanks here too. He is an MVP of comedy, and a big part of Ugly Betty's gay sensibility." On February 11, 2008, ABC picked up Ugly Betty for the 2008-09 television season, but on the day the renewal was announced, Pennette, along with fellow executive producer James Hayman, were let go. The departures of Pennette and Hayman added to the constant turnovers on the series off-camera, which has so far seen five writers having exited or been fired.