BACKGROUND: Nobiletin is a non-toxic dietary flavonoid that possesses anti-cancer properties. Nobiletin has been reported to reduce the risk of prostate cancer, but the mechanism is not well understood. In this study, we investigated the effects of nobiletin in prostate cancer cell lines PC-3 and DU-145. METHODS: Nobiletin was isolated from a polymethoxy flavonoid mixture using HPLC, cell viability was analyzed with MTS-based assays. Protein expression was examined by ELISA and western blotting. Gene expression was examined by luciferase assay. And the pathways were examined by manipulating genetic components with plasmid transfection. RESULTS: Data showed that nobiletin decreased cell viability in both prostate cell lines, with a greater reduction in viability in PC-3 cells. HIF-1alpha expression and AKT phosphorylation were decreased in both cell lines. The VEGF expression was inhibited in PC-3 but not DU-145 cells. cMyc expression was decreased in DU-145 cells. Nobiletin down-regulated NF-kappaB (p50) expression in nuclei of DU145 cells but not whole cells. It also suppressed NF-kappaB expression in both whole cells and nuclei of PC-3 cells. Increasing HIF-1alpha levels reversed nobiletin's inhibitory effects on VEGF expression, and up-regulating AKT levels reversed its inhibitory effects on HIF-1alpha expression. We speculate that AKT influences cell viability probably by its effect on NF-kappaB in both prostate cells. The effect of nobiletin on VEGF expression in PC-3 cell lines was through the AKT/HIF-1alpha pathway. CONCLUSION: Taken together, our results show that nobiletin suppresses cell viability through AKT pathways, with a more profound effect against the more metastatic PC-3 line. Due to this enhanced action against a more malignant cell type, nobiletin may be used to improve prostate cancer survival rates.