CXC chemokine receptor 4 (CXCR4) has been implicated in prostate cancer metastasis and this receptor also acts as a coreceptor for HIV-1 120-kDa glycoprotein variant IIIB (gp120-IIIB). The interaction between CXCR4 and gp120-IIIB has been shown to mediate apoptosis of both immune and endothelial cells. In this study, we have examined the effects of gp120-IIIB on hormone-refractory prostate cancer cells (PC3 and DU145) in vitro and tumor growth in vivo. Normal prostatic epithelial (PrEC) and prostate cancer cell lines were treated with gp120-IIIB with or without anti-CXCR4 antibody. Caspase expression was evaluated by real-time PCR and active caspase assays. Apoptosis was determined by flow cytometry. gp120-IIIB treatment correlated with active caspase-3 and -9 expression and apoptosis of prostate cancer cells but not PrEC cells. This effect was significantly inhibited after CXCR4 blockade. PC3 and DU145 tumor-bearing mice received intraperitoneal injections of gp120-IIIB and controls received bovine serum albumin in PBS. PC3 and DU145 tumor sizes were measured over time and excised tumors were evaluated for CD44, CD34, lymphatic endothelial cell marker LYVE-1, active caspase-3, and active caspase-9 expression by immunohistochemistry. The tumor size in mice receiving gp120-IIIB was significantly smaller than compared with tumors in control mice. This regression was associated with significant decreases in CD44, CD34, and LYVE-1 and increases in active caspase-3 and -9 expression. These results suggest that gp120-IIIB induced apoptosis in prostate cancer cells and reduced tumor-associated lymphoendothelial cells.