The Argentinean striker, who is just one goal short off the record of Gerd Muller of scoring 85 goals in a calendar year, had to be stretchered off the pitch with eight minutes remaining Wednesday night after appearing to damage his left knee in a clash with Benfica goalkeeper Artur.
He had only been on the pitch for 24 minutes when the injury happened and Barca fans will be holding their breath until the club are able to release information about the injury.
Barca coach Tito Vilanova lived up to his pre-match declarations and played a side filled with players needing match fitness and others from the B-team.
Rafinha and Thiago Alcantara shared a first team place alongside David Villa and Christian Tello, but the big surprise was Messi's absence from the starting eleven when everyone had expected to start.
Although the talented Sergi Roberto gave signs of his quality in midfield, a motivated Benfica had the best of the first half and Barca keeper Jose Pinto was Barca's best player in a first half where Benfica were in control.
More From This Section
He did well to deny former Barca B-teamer, Nolito and them saw how Rodrigo Lima hit the woodwork, before a highly motivated Nolito saw a header drift just wide.
The second half saw a different attitude from Barca, who dominated the opening 12 minutes with Tello on lightening form, before Messi got onto the pitch with 32 minutes remaining.
Messi saw a free kick saved by the keeper with 11 minutes remaining, but his best chance came after 82 minutes, when he was set through on Benfica keeper Artur, the keeper blocked his run and although Messi was able to get a shot on target, the keeper made the save and the striker had to be stretchered off the pitch.
The moment left the Camp Nou stunned, although the contact didn 't look too bad, now there will be a nervous wait until the full extent of his injury is known.
Meanwhile it was also a bad night for Benfica after Glasgow Celtic's win at home to Spartak Moscow relegated them to third place in the group and sees them miss out on a place in the next round of the competition.