A series of novel steroidal-chalcone derivates were designed and synthesized based on the molecular hybridization strategy and further evaluated for their growth inhibitory activity against three human cancer cell lines. The MTT results indicated that most compounds were apparently more sensitive to human breast cancer cells MDA-MB-231. Compounds 8 and 18 exerted the best cytotoxic activity against triple-negative MDA-MB-231 cells with the IC<sub>50</sub> values of 0.42 μM and 0.52 μM respectively, which were 23-fold increase or more compared with 5-Fu. Further mechanism studies demonstrated that compound 8 could induce cells apoptosis through regulating Bcl-2/Bax proteins and activating caspase-3 signaling pathway. Moreover, compound 8 could upregulate the cellular ROS levels which accelerated the apoptosis of MDA-MB-231 cells. In addition, interestingly, cell cycle assay showed that compound 8 could arrest MDA-MB-231 cells at S phase but not commonly anticipated G2/M phase. These evidences fully confirmed that compound 8 could be a potential candidate that deserves further development as an antitumor agent against triple-negative breast cancer.