The identification of primary molecular targets of cancer-preventive phytochemicals is essential for a comprehensive understanding of their mechanism of action. In the present study, we investigated the chemopreventive effects and molecular targets of acacetin, a flavonoid found in Robinia p seudoacacia, also known as black locust. Acacetin treatment significantly suppressed epidermal growth factor (EGF)-induced cell transformation. Immunoblot analysis revealed that acacetin attenuated EGF-induced phosphorylation of Akt and p70(S6K), which are downstream effectors of phosphatidylinositol 3-kinase (PI3-K). An immunoprecipitation kinase assay of PI3-K and pull-down assay results demonstrated that acacetin substantially inhibits PI3-K activity by direct physical binding. Acacetin exhibited stronger inhibitory effects against anchorage-dependent and -independent cell growth in cells expressing higher PI3-K activity compared with those exhibiting relatively low PI3-K activity. Binding assay data combined with computational modeling suggest that acacetin binds in an adenosine triphosphate (ATP)-competitive manner with the p110α subunit of PI3-K and interacts with Val828, Glu826, Asp911, Trp760, Ile777, Ile825, Tyr813, Ile910 and Met900 residues. Acacetin was also found to significantly reduce SK-MEL-28 tumor growth and Akt phosphorylation in vivo. Taken together, these results indicate that acacetin is an ATP-competitive PI3-K inhibitor and a promising agent for melanoma chemoprevention.