Background: ‘FufangKushen injection’ was a Chinese Traditional anticancer drug, which has been widely used to treat cancer in combination with other anticancer drugs. Objective: Our goal is to synthesize a series of novel 13-dithiocarbamates matrine derivatives using matrine (1) as the lead compound, and evaluate the biological activities of the obtained compounds. Method: The in vitro cytotoxicity of the target compounds against three human cancer cell lines (Hep3B, LM3 and BeL-7404) was evaluated. To investigate the mechanism of biological activity, cell cycle analysis was performed. Result: The results revealed that compounds 6o and 6v displayed the most significant anticancer activity against three cancer cell lines with IC50 values in the range of 3.42–8.05 µM, which showed better activity than the parent compound (Matrine). SAR analysis indicated that the introduction of a substituted amino dithiocarbamate might significantly enhance the antiproliferative activity. Conclusion: During the newly synthesized compounds, matrine analog 6v exhibited a potent effect against three human tumor cell lines. The mode of action of 6v was to inhibit the G0/G1 phase arrest. Therefore, compound 6v has been selected as a novel-scaffold lead to further structural optimizations or as a chemical probe for exploring anticancer pathways of this kind of compounds. © 2021 Bentham Science Publishers.