Abstract: This study is to investigate the anti-tumor activities of a novel cyclophosphamide derivate 4, 6-diphenyl cyclophosphamide (9b) in vivo and in vitro, and its possible mechanism of action. The inhibitory effects of 9b on human hepatoma cell line HepG₂, human breast carcinoma cell line MCF-7 and human myeloid leukemia cell line K562 were measured by MTT assay in vitro. Cell cycle distribution and apoptotic rate were evaluated by flow cytometry. To evaluate the anti-tumor effect of 9b in vivo, mouse model bearing inoculated H₂₂ tumor was established. The results indicated that 9b could inhibit the proliferation of HepG₂, MCF-7 and K562 cells in a dose and time dependent manner. The IC₅₀ values of 9b were 32.34 μmol·L^-1 to HepG₂ cells, 87.07 μmol·L^-1 to MCF-7 cells and 149.10 μmol·L^-1 to K562 cells after incubation for 48 h. The results of flow cytometry indicated that after being treated for 48 h with different concentrations of 9b, the ratios of HepG₂, MCF-7 cells at the G₀/G₁ phase and K562 cells at the G₀/G₁ phase and G₂/M phase increased significantly compared with control group, and the apoptotic rate increased with the increase of the concentration of 9b. 9b could significantly reduce tumor weight of H₂₂ solid tumor mouse model in vivo. To summarize, 9b showed significantly anti-tumor activity in vivo and in vitro, of which the mechanism might be associated with the change of cell cycle distribution and induction of tumor cell apoptosis.