Messi matched the total of 643 goals that Brazil legend Pele – one of the greatest players to grace the game – racked up in an 18-year career at Santos.
The six-time Ballon d'Or winner headed in his record-equalling goal against Valencia on Saturday and surpassed Pele's tally on Tuesday.
Having created the opener with a cross for Clement Lenglet and played a crucial role in Barca's second against Real Valladolid, Messi swept home after latching onto Pedri's clever flick in the 65th minute to make it 3-0.
As well as taking him past Pele, it was also Messi's 451st goal in LaLiga.
His effort against Valencia made the 33-year-old the first player to reach the 450 milestone for a single club in any of Europe's top five leagues.