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ZSTK474 is a novel orally applicable phosphoinositide 3-kinase-specific inhibitor that strongly inhibits cancer cell proliferation. Combination treatment using X-rays then ZSTK474 given orally for 8 days, starting 24h post-irradiation, significantly enhanced cell growth inhibition. The combined effect was also observed for clonogenic survival with continuous ZSTK474 treatment. Western blot analysis showed enhanced phosphorylation of Akt and GSK-3β by X-irradiation, whereas phosphorylation was inhibited by ZSTK474 treatment alone. Treatment with ZSTK474 after X-irradiation also inhibited phosphorylation, and remarkably inhibited xenograft tumor growth.