A series of formononetin nitrogen mustard derivatives were synthesized and evaluated in vitro for their cytotoxicity against five cancer cell lines (SH-SY5Y, HCT-116, DU-145, Hela and SGC-7901). The pharmacological results showed that many of the new derivatives displayed more potent cytotoxicity than alkeran. Furthermore, compounds 6d and 6n could induce cell cycle arrest at G2/M phase and cell apoptosis.