DFL said its supervisory board had held a meeting earlier and decided Hopfen "had different ideas about the future strategic direction of the company."
Hopfen's resignation comes amid delays to the sale of a $3.6billion stake in the Bundesliga's media rights business.
"We have achieved a lot and initiated a lot in the last few months. We have developed a viable future strategy for German professional football," Hopfen said.
Chairman of DFL's supervisory board Hans-Joachim Watzke said Hopfen had "provided important impetus for the Bundesliga."
Axel Hellmann from football club Eintracht Frankfurt and Oliver Leki from Freiburg are possible successors for the role, according to German media reports.