Messi had been called into the national squad by coach Alejandro Sabella, however he has failed to overcome the muscle problem that saw him miss Barcelona's pre-season tour of Asia and will not play a part in Wednesday's international friendly clash.
The reigning FIFA World Player of the Year struggled with a hamstring injury in the latter part of the 2012-13 season, and the Spanish giants will be hoping that further recuperation will allow him to be fit in time for the opening game of the Liga campaign.
Barca open their domestic season with a home clash against Levante on Sunday.
A statement on Barcelona's official website confirmed: "Leo Messi will not feature in the international friendly between Argentina and Italy in Rome this Wednesday.
"After undergoing tests conducted by the Argentina national team doctors, it was decided that Messi should return to Barcelona and focus on recovering from his muscle injury."
