To discover the promising antitumor agents, a series of beta-carboline derivatives with nitrogen mustard moieties were designed and synthesized. Most target derivatives showed antiproliferative activity against MCF-7 and MDA-MB-231 cells. Among them, (1-methyl-9H-pyrido[3,4-b]indol-3-yl)methyl (S)-3-(4-(bis(2-chloroethyl) amino)phenyl)-2-formamidopropanoate possessed the most potent antiproliferative activity with IC50 values of 1.79 mu M and 4.96 mu M, respectively, which were significantly higher than that of the parent compounds, and the efficacy was comparable to that of the positive control doxorubicin. More importantly, it showed weak cytotoxicity against human normal breast cell line MCF-10A (IC50 > 20 mu M), exhibiting certain selectivity. Subsequently, further mechanism exploration indicated that it induced G2/M phase cell cycle arrest and apoptosis in MDA-MB-231 cells. The DCFH-DA fluorescent probe assay and comet assay showed that this compound could cause intracellular ROS accumulation and DNA damage. In addition, it exerted potent inhibitory effect on the migration, invasion and adhesion of MDA-MB-231 cells in vitro. In short, (1-methyl-9H-pyrido[3,4-b]indol-3-yl) methyl (S)-3-(4-(bis(2-chloroethyl)amino)phenyl)-2-formamidopropanoate was considered as a promising compound for anti-breast cancer.