Defending champion Barca had only lost one competitive game all season but found itself three goals down after first-half strikes from Amandine Henry, Ada Hegerberg and Catarina Macario.
Blaugrana captain Alexia Putellas pulled one back just before the interval, but Lyon controlled the second half in Turin to ease to another European success.
Lyon required just six minutes to take the lead as Henry scored an incredible solo goal, dancing inside from the left before firing a remarkable, long-range effort into the top-right corner.
Jennifer Hermoso was denied by Christiane Endler as Barca looked to respond, but Lyon struck again when Selma Bacha crossed for Hegerberg, who headed in her 59th goal in 60 UEFA Women's Champions League games.
Hegerberg almost added a third but Sandra Panos raced out to thwart the striker, who turned provider in the 33rd minute by teeing up a simple tap-in for Macario.
Putellas reduced the deficit by volleying home Caroline Graham Hansen's right-wing centre, while Patri Guijarro hit the crossbar with an audacious strike from the halfway line after the interval.
Barca substitute Asisat Oshoala headed a golden opportunity wide and Hegerberg saw a stoppage-time volley hit the post as Lyon cruised to victory.