After beginning his acting career in regional theatre in Seattle, Washington, Jackson moved to Manhattan and was an understudy in the Broadway productions of Thoroughly Modern Millie (2002) and Aida (2003).

He next originated the role of Matthew in the workshop production of Altar Boyz (2004) for the New York Musical Theatre Festival, and was replaced by Scott Porter for the Off-Broadway run.

His mother taught Jackson, his sister, and his two brothers to sing and regularly played music by Joan Baez, Joni Mitchell, Bob Dylan, and Elvis Presley at home.

In June 2007, less than a week before the scheduled opening night, Jackson assumed the lead role of Sonny on Broadway in Xanadu, replacing James Carpinello who had been injured during rehearsal.

[15][16][17] Jackson also starred in the development workshop of The Book of Mormon by Trey Parker, Matt Stone, and Robert Lopez, who co-wrote the music for Avenue Q.

[18] In October 2009, Jackson opened on Broadway reprising the role of Woody Mahoney in the revival of the 1947 musical Finian's Rainbow at the St. James Theatre, costarring with Jim Norton and Kate Baldwin.

[19][20][21][22][23] The Wall Street Journal wrote, "The way that [Kate Baldwin] and Cheyenne Jackson sing "Old Devil Moon" is the stuff best-selling cast albums are made of.

Along with the company of Finian's Rainbow, Jackson recorded the Broadway revival cast album in early December 2009, with a release date of February 2, 2010.

[25] In September 2011, Jackson joined Morgan Freeman, Ellen Barkin, Matt Bomer, Christine Lahti, and John Lithgow as part of the Broadway-premiere cast of Dustin Lance Black's new play 8, directed by Joe Mantello.

In June 2012, Jackson starred opposite Krysten Ritter in Neil LaBute's The Heart of The Matter produced by MCC Theater at the Lucille Lortel Theatre.

In the 2012–2013 Broadway season, Jackson, Henry Winkler, Alicia Silverstone, Daniel Breaker, Jenni Barber and Ari Graynor starred in David West Read's play The Performers which started previews on October 23, 2012, at the Longacre Theatre.

[27] In 2019, Jackson made his Hollywood Bowl debut playing the roles of the Wolf/Cinderella's Prince in the show Into the Woods opposite Sutton Foster, Sierra Boggess, Patina Miller, Skylar Astin, Chris Carmack, and Shanice Williams.

[31] He played Michael Minetti, who is hired by retiree Lily Harrison (Gena Rowlands) to give her private dance lessons at her home for six weeks.

Jackson also portrayed Dustin Goolsby, the coach of "Vocal Adrenaline", the main rival of "New Directions", on the second season of FOX's show Glee.

[34] In 2008, Jackson played series lead Sebastian Kinglare for the Lifetime Television pilot Family Practice opposite Anne Archer and Beau Bridges.

In 2010, Jackson filmed a sitcom pilot for ABC-TV called It Takes a Village, co-starring Leah Remini, which was not picked up for the fall 2010 season.

Jackson portrayed Scout Master Steve, a competing love interest for Portia de Rossi's character Lily Munster; the show also stars Eddie Izzard and Jerry O'Connell.

He also appeared in the tenth episode of Season 2 of Sense8 as Blake Huntington, an award-winning actor who is cast opposite Miguel Ángel Silvestre's Lito Rodríguez in a Hollywood film.

[38][39] Since 2020, Jackson costars as Max in the Fox comedy series Call Me Kat opposite Mayim Bialik and Kyla Pratt.

[42][43] In March 2009, Jackson made his nightclub debut at Feinstein's at Loews Regency with a sold-out one-man show titled Back to the Start.

"[52] On December 31, 2012, Jackson reprised Music of the Mad Men Era at The Kennedy Center Concert Hall with 96 members of the National Symphony Orchestra and Nina Arianda.

On June 3, 2016, Jackson released Renaissance,[7] an album on the PS Classics record label, adapted and expanded from his solo concert Music of the Mad Men Era.

In 2008, he was named "Entertainer of the Year" by Out and appeared alongside Gus Van Sant, Katy Perry, and Sam Sparro on the cover of the magazine's commemorative 100th issue in December.

In April 2010, he appeared on the cover of Canadian fab magazine under the title "Cheyenne Jackson: I Love New York" with photographs by Mike Ruiz.