Brefeldin A has a wide range of anticancer activity against a variety of tumor cells. Its poor pharmacokinetic properties and significant toxicity seriously hinder its further development. In this manuscript, 25 brefeldin A-isothiocyanate derivatives were designed and synthesized. Most derivatives showed good selectivity between HeLa cells and L-02 cells. In particular,6exhibited potent antiproliferative activity against HeLa cells (IC50= 1.84 μM) with no obvious cytotoxic activity to L-02 (IC50> 80 μM). Further cellular mechanism tests indicated that6induced HeLa cell cycle arrest at G1 phase. Cell nucleus fragmentation and decreased mitochondrial membrane potential suggested6could induce apoptosis in HeLa cells through the mitochondrial-dependent pathway.