ZSTK474 is a novel phosphatidylinositol 3-kinase (PI3K) inhibitor, which strongly inhibits cancer cell proliferation. Phosphoinositide 3-kinase (PI3K) is a potential target in cancer therapy. Inhibition of PI3K is believed to induce apoptosis. ZSTK474 effectively inhibited the growth of human cancer xenografts in vivo. ZSTK474 treatment suppressed the expression of nuclear cyclin D1 and Ki67 and also the expression of phospho-Akt, both of which are hallmarks of proliferation. We demonstrate that ZSTK474 induces G1arrest along with tumour suppression. Moreover, ZSTK474 suppresses the tumour growth without inducing apoptosis. Interestingly, long-term (3-month) administration of ZSTK474 maintained such increase in G1 cells and tumour suppression. ZSTK474 exerts its in vivo antitumour efficacy via G1 arrest but not via apoptosis as long as it is administered.