OBJECTIVE To evaluate the inhibitive effect of C-21 steroidal glycosides from the root of Cynanchum auriculatum (CGB) on rat glioma C6 cells. METHODS C6 cells were treated with CGB for 24, 48,72 h at concentration of 30, 60, 120 mg/L, respectively. MTT assay was used for evaluating cell viability; fluorescence-activated cell sorting analysis after Annexin V/propidium iodide staining or single propidium iodide staining was used to test cell apoptosis and cell cycle. RESULTS CGB at 30, 60, 120 mg/L concentration-dependently decreased C6 cell viability (P<0.001). CGB at 60 and 120 mg/L induced C6 cell apoptosis and cell cycle arrest. The fraction of G0/G1 cells was increased (P<0.05) and that of S phase cells was decreased (P<0.01). CONCLUSION CGB can inhibit the growth of rat glioma C6 cells, and induce apoptosis and G0/G1 cell cycle arrest.