Thielen injured his left ankle on the Vikings' first offensive series when he was tackled by Lions cornerback Jerry Jacobs after catching a screen pass on third-and-9.
The wideout fell to the ground and ripped his helmet off before being tended to by athletic trainers. After a short visit to the medical tent on the sideline, Thielen tested out his injured ankle behind Minnesota's bench but never returned to the field. He was initially listed as questionable to return but ruled out at the start of the third quarter.
Thielen will undergo testing on Monday to determine how much time he will miss.
Without Thielen in the game, Vikings wide receiver Justin Jefferson reached a career-high 182 yards receiving on 11 catches and scored on a 3-yard touchdown late in the fourth quarter to temporarily give the Vikings a 27-23 lead. The Lions then marched down the field on a 14-play drive that spanned 75 yards in 1:50 to lead Detroit to a walk-off win.
The Vikings (5-7) host the Pittsburgh Steelers on Thursday night to kick off Week 14.