Kightlinger was born and raised in Jamestown, New York. She attended Southwestern High School and then Emerson College.
After touring as a stand-up comic, Kightlinger got her break writing for the television sitcom Roseanne. She later wrote for Dennis Miller Live and for the 20th season (1994–1995) of Saturday Night Live, on which she was also a featured performer and cast member. Among the celebrities she impersonated were Brooke Shields, Marcia Clark, Paula Poundstone, Mary Hart, and Connie Chung.
Kightlinger worked as a writer and consulting producer on the sitcom Will & Grace. She performed a recurring guest role on the series as Sheila, a nurse at a fertility clinic. Kightlinger was a correspondent on The Daily Show in 1998. She appeared regularly on the HBO sitcom Lucky Louie. She was a writer and cast member on Roseanne’s short-lived sketch comedy series Saturday Night Special on Fox.
In 2006, Kightlinger created, wrote, executive produced and starred in The Minor Accomplishments of Jackie Woodman, a comedy series about two best friends maneuvering through the not-so-glamorous side of Hollywood. The series aired on IFC from August 4 of that year through September 23, 2007.
When asked how much of the series was based on her personal experience, Kightlinger responded: I think like 85% and then the other 15 is the experience of a woman. We usually cull a lot of stories from what happened to us and our friends, so I’d say a lot of it is. I always feel like rejection is my petrol. That’s what keeps me going. Kightlinger has had two stand-up comedy specials on HBO and six on Comedy Central.
In 2001, she made a short film, Dependable People, which won both the Black Maria Film Festival Director's Citation (Honorable Mention) and the International Festival of Cinema and Technology Best New Director Award in 2002. It was released on the DVD "Celebrity Mix" with other short films in 2006.
In 2003, she directed her first documentary, Sixty Spins Around the Sun, which chronicles the New York City street movement to repeal the Rockefeller Drug Laws. It focuses on political satirist turned activist Randy Credico and his fight to repeal the laws. The film follows Credico to Tulia, Texas during a racially motivated drug bust, which is the subject of an upcoming film starring Halle Berry.
Included in this documentary are stand-up comics Larry David, Colin Quinn, Don Gavin, Vanessa Hollingshead and Nick DiPaolo. It won Best Documentary at the 2003 Empire State Film Festival, 2003 Boston International Film Festival, and 2005 Beverly Hills Film Festival (Jury Award). Kightlinger appeared in Shallow Hal in 2004. In 2005 she starred in the short film Dysenchanted, directed by Terri Edda Miller, which received critical acclaim.
Kightlinger's book Quick Shots of False Hope was published in 1999. The New York Times Book Review described it as "funny and disturbing", "memorable", and an "idiosyncratic and darkly comic debut." As of 2006, she was adapting the book for film.
Kightlinger has written several shorts for Funny or Die and Atom.com, including “American Heroine,” in which she starred, and “Roy Fabcock: Legendary Lover.” (2010).