The museum announced that Barbara Buhler Lynes resigned Friday but declined to say why.
The announcement comes on opening day of a new exhibit that highlights the importance of O'Keeffe's camping and rafting trips through the Southwest and the inspiration they provided her art.
Lynes could not immediately be reached for comment but said in a statement issued by the museum that she enjoyed her time there and looks forward to pursuing new projects.
Lynes is considered the world's leading authority on the life and art of the late American modernist painter.
She became the museum's first curator in 1999. She oversaw more than 30 exhibitions and organized several symposiums that attracted scholars from around the country.