Abstract Aberrant overexpression of antiapoptotic members of the Bcl-2 protein family, including Bcl-2 and Bcl-XL, contributes to malignant transformation and subsequent resistance to traditional chemotherapeutics. Thus, these proteins represent attractive targets for novel anticancer agents. The small molecule, gossypol, was initially investigated as a contraceptive agent, but subsequently has been shown to possess anticancer properties in vitro and in vivo. Recently gossypol has been found to bind to Bcl-XL and, with less affinity, to Bcl-2. Here we investigate the ability of the (−) enantiomer of gossypol, (−)-gossypol, to overcome the apoptosis resistance conferred by Bcl-2 or Bcl-XL overexpression in Jurkat T leukemia cells. (−)-Gossypol potently induced cell death in Jurkat cells overexpressing Bcl-2 (IC50, 18.1 ± 2.6 μmol/L) or Bcl-XL (IC50, 22.9 ± 3.7 μmol/L). Vector-transfected control cells were also potently killed by (−)-gossypol (IC50, 7.0 ± 2.7 μmol/L). By contrast, the chemotherapy drug etoposide only induced efficient killing of vector-transfected cells (IC50, 9.6 ± 2.3μmol/L). Additionally, (−)-gossypol was more efficient than etoposide at inducing caspase-3 activation and phosphatidylserine externalization in the setting of Bcl-2 or Bcl-XL overexpression. (−)-Gossypol-induced apoptosis was associated with Bak activation and release of cytochrome c from mitochondria, suggesting a mitochondrial-mediated apoptotic mechanism. Moreover, (−)-gossypol treatment of isolated mitochondria purified from Bcl-2-overexpressing cells also resulted in cytochrome c release, indicating a possible direct action on Bcl-2 present in the mitochondrial outer membrane. Taken together, these results suggest that (−)-gossypol is a potent and novel therapeutic able to overcome apoptosis resistance by specifically targeting the activity of antiapoptotic Bcl-2 family members. (−)-Gossypol may be a promising new agent to treat malignancies that are resistant to conventional therapies.