Greg Baker, 62, resigned effective Tuesday, giving no reason for his departure, The Kansas City Star reported.
"We have accepted his resignation," museum board Chairman Pellom McDaniels said in a statement. "The board wishes him well in his future endeavors."
The 20-year-old museum has been a prime tourist attraction for Kansas City, but its financial fortunes have seen ups and down.
A former administrator in the Kansas City Aviation Department, Baker was given the $115,000-a-year museum job on an 8-to-7 board vote.
Some board members had lobbied for Bob Kendrick, the museum's longtime marketing director and friend of baseball legend Buck O'Neil.
A former board member said Baker's resignation came as a surprise.
Mark Bryant said he had opposed the hiring because Baker "did not have the long-established relationships the other candidate (Bob Kendrick) had with the media, our funders, with our public or our membership."
But Bryant said he was "grateful for his service and confident the Negro Leagues Baseball Museum will continue to be our nation's repository of this important history."