Palicki was born in Toledo, Ohio, to Nancy and Jeff Palicki. She graduated from Whitmer High School in Toledo in 2001. She played basketball and ran track in high school and was named runner-up for homecoming queen.
Palicki stands 5'11" (180 cm) tall. Her nickname is "Annie."
Palicki is currently dating actor Alan Tudyk.
Palicki appeared in the Aquaman-based WB pilot Aquaman as the evil Siren, but the pilot was not picked up for series by the CW network, a result of The WB and UPN network merger, which occurred while the pilot was being filmed. She also appeared in the first 3 seasons of NBC's much acclaimed Friday Night Lights.
Palicki also starred in Supernatural as Jessica Moore, Sam Winchester's doomed girlfriend, killed by the demon, in its pilot episode.