Using pregnenolone as starting material, 16 steroidal derivatives Using pregnenolone as starting material, 16 steroidal derivatives containing benzimidazole heterocycle were designed and synthesized by suitable modification at C17 side chain of pregnenolone, their structures were characterized by IR, 1H NMR、13C NMR and HRMS. The antiproliferative activity of the target compounds in vitro was evaluated against KB, HeLa, HepG, CNE-2, BT474 and SKOV3 cancer cells by MTT method. The results show that some compounds possess distinct antiproliferative activities against tested cells. Among the active compounds, compound 5c and 6c show better selective activities against SKOV3 cells with IC50 values of 15.4±3.8 and 9.2±0.5 μmol/L, respectively. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.