Resistance to chemotherapy is a common problem encountered in the treatment of head and neck squamous cell carcinoma (HNSCC). Chemoresistant HNSCC tumors frequently overexpress antiapoptotic proteins, such as Bcl-x(L). (-)-gossypol, the negative enantiomer of a cottonseed polyphenol, binds to Bcl-x(L) and was recently been shown to inhibit HNSCC proliferation in vitro. In this study, we assessed the in vivo efficacy of (-)-gossypol in an orthotopic xenograft model of HNSCC, using two human HNSCC cell lines with high Bcl-x(L) expression levels. Both produced tumors in a murine floor-of-mouth model that mimics human HNSCC, exhibiting growth and invasion into adjacent tissues. Mice were randomized into three groups: vehicle control and two daily intraperitoneal (-)-gossypol treatment groups (5 and 15 mg/kg). Tumors were measured twice weekly. In the control group, tumors grew progressively, whereas in (-)-gossypol treatment groups, tumor growth was significantly suppressed. The mitotic rate in tumors from (-)-gossypol-treated animals was significantly lower than that in controls, and an increase in the percentage of apoptotic cells was observed in treated tumors versus controls. Residual tumors remained growth-suppressed for 2 weeks after cessation of (-)-gossypol treatment. Our results demonstrate that (-)-gossypol can inhibit tumor growth in an orthotopic model of aggressive HNSCC.