Hayden Panettiere began modeling at the age of five months. She then began appearing in commercials at 11 months, first appearing in an advertisement for Playskool. She landed a role as Sarah Roberts on the ABC soap opera One Life to Live (1994- 1997) and later as Lizzie Spaulding on the CBS soap opera Guiding Light when she was seven years old (1996 - 2000). While on Guiding Light, Panettiere's character Lizzie battled leukemia. The show received a Special Recognition Award from the Leukemia and Lymphoma Society for bringing national awareness of the disease to the attention of daytime viewers.

Panettiere appears as Claire Bennet in the NBC series Heroes as a high school cheerleader with regenerative healing powers. Due to her role on Heroes, she became a regular on the science fiction convention circuit, invited to attend conventions around the world in 2007, including New York Comic-Con and Fan Expo Canada. In regards to the roles she has played, Panettiere admitted that options are sometimes limited because "people look at [her] as either the 'popular cheerleader' type or just 'the blonde.'"

She has appeared in over a dozen full-length feature films, as well as several made-for-TV movies. She provided the voice for Dot in the Pixar animated movie A Bug's Life.  She also played the role of Coach Yoast's daughter, Sheryl, in the 2000 Disney film Remember the Titans. In addition, she appeared as the voice of Kairi in the Kingdom Hearts series of video games for Playstation 2. Panettiere appeared on FOX's Ally McBeal as Ally McBeal's daughter, had a recurring guest role on Malcolm in the Middle, and guest starred in Law & Order: SVU. She starred in Bring It On: All or Nothing as a cheerleader and had a supporting role as Adelaide Bourbon in the recently released independent film Shanghai Kiss. She also appeared in the Disney Channel Original Movie Tiger Cruise playing the main role of a Navy brat.

She appears in the upcoming drama film Fireflies in the Garden as a younger version of Emily Watson's character, Jane Lawrence. In June 2007, she signed with the William Morris Agency after previously being represented by United Talent Agency.

In early 2007, Panettiere appeared on the syndicated Ashton Kutcher-produced show, Punk'd. The appearance was engineered by her mother and involved a male "fan" discussing her work with her, instigating a jealous reaction from the man's spouse.