Gossypol is a naturally occurring polyphenolic compound extracted from cotton plants. Recent studies revealed that gossypol is a non-peptidic small molecule inhibitor of Bcl-2/Bcl-xl. The aim of the present study was to investigate the induction of apoptosis and antitumor effects of gossypol acetate in multiple myeloma and the possible mechanism(s) of action. Our results showed that gossypol acetate resulted in a dose- and time-dependent inhibition of multiple myeloma cell proliferation, with an IC50 value to both U266 and Wus1 cells at 2.4, 2.2 µM at 48 h after treatment. Gossypol acetate effectively induced the apoptosis of multiple myeloma cells as demonstrated by typical morphological changes, DNA ladder formation and increase in the percentage of cells in subdiploid peak. Furthermore, colorimetric assays showed activation of both caspase-3 and caspase-9. Bcl-2 and Bcl-xl expression was decreased by 86.5±1.2% and 35.9±3.6%, respectively, after treatment with gossypol acetate at 25 µmol/l for 24 h. Preliminary studies in vivo showed that a growth inhibition (T/C) of 30.9% (gossypol acetate 40 mg/kg) was obtained in Balb/C mice bearing Wus1 cells. In addition, there was no body weight loss for the treated group in comparison with the vehicle mice. Our results demonstrated that the potent inhibitor of Bcl-2 and Bcl-xl gossypol acetate had significant antiproliferative and antiapoptotic effects on multiple myeloma cells in vitro and in vivo. Gossypol acetate may represent a promising new anticancer agent with a novel molecular mechanism and warrants further investigation as a single agent, or in combination with other chemotherapeutics, for human multiple myeloma with Bcl-2 overexpression.