Chronic myelogenous leukemia (CML) is a malignant hematological disorder mainly caused by the Bcr-Abl tyrosine kinase. While Bcr-Abl inhibitors including Imatinib showed antitumor efficacy on many CML patients, resistance was frequently reported in recent years. Therefore, novel drugs for CML are still expected. ZSTK474 is a specific phosphatidylinositol 3-kinase (PI3K) inhibitor that we identified. In the present study, the efficacy of ZSTK474, alone or in combination with Imatinib, on K562 CML cells as well as on its multidrug resistance counterpart K562/A02 cells, was investigated. ZSTK474 inhibited the cell proliferation with an IC50 of 4.69 μM for K562 and 7.57 μM for K562/A02 cells, respectively. Treatment by ZSTK474 resulted in cell cycle arrest in G1 phase, which might be associated with upregulation of p27, and downregulation of cyclin D1. ZSTK474 also inhibited phosphorylation of Akt and GSK-3β, which might be involved in the effect on the above cell cycle-related proteins. Moreover, combination of ZSTK474 and Imatinib indicated synergistic effect on both cell lines. In conclusion, ZSTK474 exhibited antileukemia activity alone, and showed synergistic effect when combined with Imatinib, on CML K562 cells as well as the multidrug resistant ones, providing a potential therapeutic approach for CML patients.