The Barcelona star played the full 90 minutes of friendly but it was confirmed after the game that he will now leave the squad and return to his club to recover.
Messi was making his first international appearance since last summer’s World Cup in Russia.
The five-time Ballon d’Or winner missed Argentina’s last six fixtures since they crashed out of the tournament to France in the last-16.
Argentina had several issues during the World Cup and were lucky to even make it out of the group stage.
And their issues don’t look to have been resolved after the disappointing loss to fellow South American’s Venezuela.
Newcastle striker Salomon Rondon scored on six minutes before Jhon Murillo doubled the lead just before half-time.
Lautaro Martinez pulled a goal back for Argentina, but Josef Martinez added Venezuela’s third with a penalty.
Barcelona will be hoping that Messi’s injury is not serious as they prepare for the season run-in.
The Spanish champions already look to have sealed La Liga once more and they are into the final of the Copa del Rey.
And the Catalans have been drawn to play Manchester United in the Champions League quarter-final.
The first leg of the contest takes place on April 10 at Old Trafford.
Messi’s injury scare comes after:
Before that Barca play rivals Espanyol on March 30 in La Liga before they travel to Villarreal on April 2.
Messi is once again the club’s top scorer this season as he continues to defy logic.
The 31-year-old has netted 39 goals in 37 games in all competitions this season.