The results of the present study revealed for the first time the possibility to use a combination of mitoxantrone with gefitinib and cyclopamine for inhibiting the growth of epidermal growth factor (EGF), sonic hedgehog- (SHHNp), and serum-stimulated androgen-sensitive LNCaP-C33 and androgen-independent (AI) LNCaP-C81, DU145 and PC3 prostate cancer (PC) cells. The supra-additive anti-proliferative effects of drugs were mediated via a blockade of the PC3 cells in the G(1) and G(2)M phases of the cell cycle. Importantly, the combination of mitoxantrone plus gefitinib and/or cyclopamine also caused a higher rate of apoptotic death of PC cells including enriched fraction of CD44(high) PC3 cell subpopulation as compared to the individual agents or bi-combination of drugs. The cytotoxic effects induced by mitoxantrone, gefitinib and cyclopamine on PC3 cells appear to be at least partly mediated through the depolarization of the mitochondrial membrane, release of cytochrome c into the cytosol, hydrogen peroxide production and activation of caspase cascades. These findings indicate that the simultaneous blockade of EGF-EGFR and sonic hedgehog tumorigenic signaling cascades may represent a promising strategy for improving the efficacy of current mitoxantrone-based therapies against incurable AI and metastatic PCs in the clinics.