Padalecki was born in San Antonio, Texas to Sharon L. (Kammer), a high school English teacher, and Gerald R. Padalecki, a tax supervisor and accountant. His paternal grandfather was Polish. He has an older brother, Jeff, and a younger sister, Megan. He attended James Madison High School in San Antonio and was named a candidate for the 2000 Presidential Scholars Program. In 1998, Padalecki and his partner Chris Cardenas won the National Forensic League national championship in Duo Interpretation.

Padalecki won Fox TV's 1999 "Claim to Fame Contest"; he subsequently appeared at the Teen Choice Awards, where he met his current manager. Before graduating from high school in 2000, he moved to Los Angeles, California to pursue an acting career, although he had originally planned to attend the University of Texas.

Padalecki's first credit was a minor role in the 1999 film, A Little Inside. In 2000, he was cast as Dean Forester on the successful series Gilmore Girls, a role he played until 2005. Throughout the early 2000s, he also appeared in several made-for-television films, including Silent Witness (2000), Close to Home (2001), A Ring of Endless Light (2002) and Young MacGyver (2003).

Padalecki had an uncredited part in 2003's comedy Cheaper by the Dozen. In 2004, he appeared in the Mary-Kate and Ashley Olsen-led comedy New York Minute, and also had a role in the thriller Flight of the Phoenix. In 2005, Padalecki starred opposite Elisha Cuthbert, Chad Michael Murray and Paris Hilton in the horror film House of Wax, and appeared in another horror film, Cry Wolf, on the set of which he met his longtime girlfriend, co-star Sandra McCoy.The same year, Padalecki was cast as Sam Winchester on the WB series, Supernatural, and gained a fan base after the show's success. Supernatural was renewed for a second season, which began airing on September 28, 2006 on The CW. The show was later renewed for a third season to air on October 4, 2007 at 9pm.

 Padalecki most recently featured as host in an advertisement for MTV's upcoming horror reality series, ROOM 401. He hosts the series, produced by Ashton Kutcher.