Messi was reduced to the role of spectator at the Etihad Stadium, where goals from Ever Banega and Manuel Lanzini ensured the win for Jorge Sampaoli's men.
The Barcelona superstar has been in typically free-scoring form for the Blaugrana, finding the net eight times in his last six games, with his tally for the season up to 35.
And that run looks even more impressive following Messi's admission of his injury problems, though he is hopeful of being fit to face Spain on Tuesday.
"I have been suffering from this hamstring injury for some time," Messi told TyC Sports before the game.
"I always want to play, but the road to the World Cup is still long. We decided to wait for this game [against Italy] to rest but I'm optimistic about playing against Spain."
Head coach Jorge Sampaoli added in his post-game media conference: "We had planned the game with Leo Messi in the starting line-up, but he had an injury and we preferred him not to take part."
Angel Di Maria, though, will miss the meeting with Spain in Madrid due to a problem with his right thigh.
The Paris Saint-Germain midfielder was replaced by Diego Perotti in the 64th minute of Friday's encounter and the Argentine Football Association (AFA) later confirmed he will sit out the game at the Wanda Metropolitano.