Objective To investigate the effects of PTD4-GFP-Apoptin protein on proliferation inhibition and apoptosis-inducing of different types of leukemia cells. Methods Genetic engineering was used to restructure a carrier containing PTD4-GFP-Apoptin gene, and MTT was applied to detect the expressed PTD4-GFP-Apoptin fusion protein and its effect on the leukemia cell proliferation. Flow cytometry (FCM) was used to detect the effects on cell apoptosis. Results MTT cell proliferation inhibitory experiment showed that PTD4-GFP-Apoptin had different degree of proliferation inhibition on different types of leukemia cells;furthermore, the inhibitory effect presented positive correlation with time and concentration. FCM showed that PTD4-GFP-Apoptin had apoptosis-inducing effect on HL-60 cells, and the apoptotic rate had significant difference compared with the control group (P <0.05). Conclusions PTD4 can carry large proteins to penetrate the cell membrane, and PTD4-GFP-Apoptin may produce the inhibiting proliferation in vitro for a variety of leukemia cells. Apoptin can induce tumor cell apoptosis without affecting normal cells, which might become a new agent for the clinical treatment of leukemia.