Stamkos had the "C" on his jersey when he skated out to face the Buffalo Sabres in his first game since Nov. 11. The 24-year-old center had been sidelined since breaking his right tibia in a game against the Boston Bruins.
St. Louis, who had been captain since the start of this season, was traded to the New York Rangers on Wednesday.
"Steven Stamkos was destined to be the leader of our team," Lightning coach Jon Cooper said in a statement. "The fact that it's happening now just puts us one step closer to achieving our ultimate goal, and that is trying to win the Stanley Cup."
Defenseman Eric Brewer will serve as a full-time alternate captain, the Lightning said, filling Stamkos' previous position. Defenseman Matt Carle and forward Nate Thompson will continue to share an alternate designation, but will change to Carle wearing the "A" at home and Thompson wearing it on the road.
Forward Ryan Callahan, the former captain of the Rangers acquired in the trade, made his debut for the Lightning in the 3-1 loss at Tampa Bay Times Forum.