The Argentine drew level with his legendary compatriot as he struck for Barcelona against Real Madrid in the sides' Liga clash at the Santiago Bernabeu on Saturday
Messi netted after 17 minutes after Karim Benzema had given Real Madrid an early lead.
The Argentine had failed to score in the teams' recent Copa del Rey semi-final series, which Madrid won 4-2 on aggregate thanks to a 3-1 victory at Camp Nou on Tuesday, but he was on target this time to draw level with his compatriot, now Real's honorary president, on 18 strikes in the famous fixture.
Di Stefano hit 18 goals for Madrid against Barca in 30 games between 1953 and 1964, but Messi has drawn level with that total in just 25 matches.
Leo, who is still only 25, is also the last player to hit a hat-trick in the fixture, having netted all three of Barca's goals in the 3-3 draw at Camp Nou in 2006-07.