Ganoderic acid Mk (GA-Mk), a triterpenoid acid, was isolated from the mycelia of Ganoderma lucidum, and no biological activity of GA-Mk has ever been reported. In this work, we investigated the effect of GA-Mk on the cell proliferation and apoptosis in HeLa cells. The MTT results demonstrated that GA-Mk displayed interesting cytotoxicity toward various human cancer cell lines. Bromodeoxyuridine (BrdU) incorporation assay showed that GA-Mk had a dose-dependent inhibitory effect on proliferation of HeLa cells. The flow cytometry analysis indicated that the treatment of HeLa cells with GA-Mk increased the rate of early and late apoptotic cells in a dose-dependent manner. Furthermore, GA-Mk inducing apoptosis was in association with the burst of intracellular reactive oxygen species (ROS), the decrease of the mitochondrial membrane potential and the increase of caspase-3 and caspase-9 activities. These results demonstrated that GA-Mk was efficiently anti-proliferative and could induce apoptosis of HeLa cells by mitochondria-mediated pathway, and it may serve as a promising candidate for the treatment of cervical cancer.