Objective To evaluate the anti-tumor activity and possible mechanism(s) of Platycodin D (PD), a major saponin derived from Platycodin grandi?orum, in human prostate cancer cell lines. Methods Human prostate cancer cell lines (PC3, DU145 and LNCaP cells), and the non-malignant human prostate epithelial cell line, RWPE-1, were exposed to various concentrations of PD to evaluate its cytotoxicity in vitro (via the MTT assay). Cell cycle analysis was indicated by PI staining with ?ow cytometry. Cell cycle-related proteins levels were assessed by Western blotting. Results PD exerted cytotoxicity against three prostate cancer cell lines, PC3 cells, DU145 cells, and LNCaP cells, with half-maximal inhibitory concentrations (IC50) in the range of 11.17 to 26.13 μmol/L. The PC3 cells were the most sensitive to PD. After being treated with PD for 48 hours, the PC3 cells were arrested in the G2/ M phase, and the DU145 and LNCaP cells were arrested in the G0 /G1 phase. Western blotting analysis indicated that PD exposure decreased the levels of cell cycle-related proteins, including E2F1, CDK2, CDK4, CDK6, CyclinD1, Cdc2, CyclinB1. Conclusions PD exhibits signifcant inhibitory activities against prostate cancer cells, which provides a basis for future development of human prostate cancer chemotherapy.