Objective To study the effect of different concentrations of juglone on the proliferation and apoptosis of human cervical cancer cells (HeLa), and explore the anti-tumor effect of juglone in vitro. Methods HeLa cells were cultured in vitrofor 24 h and then divided into control group and juglone-treated group. The cells in juglone-treated group were cultured again with 10, 20, 50, 100 and 200μmol/L juglone, respectively, for 24h. The morphological changes in HeLa cells were observed with inverted microscope. The proliferation of HeLa cells was assessed detected by methyl thiazolyl tetrazolium (MTT) assay. The cell cycle and apoptosis were observed by flow cytometry (FCM). The expression of caspase-3 in HeLa cells was determined by caspase-3 colorimetric assay kit. Results Under morphological observation, it was found that different concentrations of juglone led to change in cell shape. MTT assay showed that juglone significantly inhibited the growth of HeLa cells in a dose-dependent manner (P<0.05 or P<0.01). FCM assay indicated that the percentage of the cell cycles arresting at G2/M phase after treatment with above concentrations of juglone for 24 h were increased to 12.92%, 16.23%, 23.64%, 34.22% and 52.64% from 7.5%, respectively (P<0.05 or P<0.01). The caspase-3 activity in juglone-treated HeLa cells remarkably increased in a dose-dependent manner as compared with control group. Conclusion Various concentrations of juglone can inhibit the proliferation and induce the apoptosis of HeLa cells in a dose-dependent manner, implying that juglone may have anti-tumor effect.