Betulinic acid (BA), a potential anticancer compound, could induce apoptosis in human cervical cancer (HeLa) cells, but its mechanism has yet to be fully elucidated. The present study was focused on deciphering the detailed molecular mechanism of BA-induced apoptosis. In the present study, results indicated that BA was highly effective against HeLa cells via induction of time-dependent apoptosis, and the authors demonstrated that the BA treatment acted through downregulating a phosphatidylinositol 3-kinase (PI3K) subunit and suppressing the Akt phosphorylation at Thr308 and Ser473 after increasing the generation of intracellular reactive oxygen species. Then, BA induced cell cycle arrest at the G0/G1 phase, which was consistent with the cell cycle-related protein results in which BA significantly enhanced the expression of p27Kip and p21Waf1/Cip1 in HeLa cells. This target-specific inhibition was associated with mitochondrial apoptosis, as reflected by the increased expression of Bad and caspase-9, the generation of reactive oxygen species (ROS) and the decline in mitochondrial membrane potential. Moreover, preincubation of the cells with glutathione (antioxidant) blocked the process of apoptosis, prevented the phosphorylation of downstream substrates. These results established that ROS acted as a key factor to effect apoptosis by BA treatment in HeLa cells. Therefore, these findings demonstrated that BA induced apoptosis in HeLa cells by downregulating the expression of PI3K/Akt signaling molecules via ROS, and triggering a mitochondrial pathway.