Melittin (MEL), a major polypeptide in bee venom (BV), is known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in MEL-induced apoptosis have not been fully elucidated, especially in human leukemic cells. In the present study, we report that MEL induces apoptosis in leukemic U937 cells through downregulating Akt signal pathways. Furthermore, MEL-induced apoptosis was accompanied by downregulation of Bcl-2 and activation of caspase-3. The induction of apoptosis also was accompanied by the downregulation of the inhibitor of apoptosis protein (IAP) family proteins. Treatment of U937 cells with the caspase-3 inhibitor, z-DEVD-fmk, was capable of significantly restoring cell viability in MEL-treated cells. Additionally, the caspase-3 mediated apoptotic response was significantly attenuated in Bcl-2-overexpressing U937 cells treated with MEL. These results indicate that downregulation of Bcl-2 plays a major role in activation of caspase-3 following MEL exposure. MEL also triggered downregulation of Akt. LY294002 (an inhibitor of Akt) significantly decreased cell viability and increased the proportion of cells with sub-G1 phase DNA content. The results indicated that key regulators in MEL-induced apoptosis in human leukemic U937 cells include Bcl-2 and caspase-3, which are controlled through the Akt signaling pathway.