This study was aimed to explore the inhibition effect and mechanism of hedyotis diffusa wild injection (HDI) on leukemia cell line (HL-60) in vitro. The leukemia cell line HL-60 was used as target cells. The inhibitory effects of HDI on proliferation of HL-60 cells were observed by MTT assay. The positive rate of cell apoptosis and the surface marker of granulocytic differentiation (CD33 and CD15) were measured by flow cytometry. The expressions of anti-apoptosis related gene (survivin and bcl-2) were detected by RT-PCR. The results showed that the growth of HL-60 cells was inhibited by higher concentration of HDI (3.12 - 12.5 ml/L) and inhibited obviously in dose-dependent manner (p < 0.05 or p < 0.01), but not suppressed by low concentration of HDI (1.56 ml/L) in liquid culture system (p > 0.05). The FCM and DNA Ladder results showed that the phenomenon of typical apoptosis did not detected after HL-60 cells were treated with the different concentrations of HDI for 24, 48 and 72 hours respectively. After HL-60 cells were treated with HDI (1.56, 3.12, 6.25 and 12.5 ml/L) for one week, the expression level of CD15 surface marker was all enhanced obviously. When treated with HDI (6.25 ml/L) for 3 weeks, the expression levels of survivin and bcl-2 gene were also decreased obviously by 60% and 44% respectively. It is concluded that HDI can inhibit HL-60 cells in the presence of its higher concentrations. The mechanisms of HDI may induce HL-60 cells differentiation, and suppress the expression of anti-apoptosis related gene (survivin or bcl-2) to inhibit the growth of HL-60 cells.