Leukemia is currently one of the most deadly diseases. Ginseng has been used in Asian countries for the treatment and prevention of various diseases, including leukemia, but the molecular mechanism of its antileukemia activity has not been well defined. The aim of this study was to explore the effect of 20-(s)-ginsenoside Rg3 on apoptosis in human leukemic U937 and HL-60 cells and the underlying mechanism. We found that 20-(s)-ginsenoside Rg3 reduced cell viability and induced apoptosis in U937 and HL-60 cells. The induction of apoptosis was accompanied by the downregulation of PI3K/Akt family proteins. Moreover, we observed that 20-(s)-ginsenoside Rg3 treatment resulted in activation of caspase-3 and caspase-9. Taken together, our findings suggest for the first time that 20-(s)-ginsenoside Rg3 can promote apoptosis in U937 and HL-60 cells, at least partly through the downregulation of PI3K/Akt family proteins. Moreover, the triggering of caspase-3 and caspase-9 activation mediated apoptotic induction. All these findings collectively demonstrate that the natural compound 20-(s)-ginsenoside Rg3 effectively induces apoptosis in human leukemic cells, which suggests that this compound may play a role in future therapies for leukemia.