Abstract: A series of novel 4-substituted-3-nitrobenzamide derivatives were designed and synthesized. The structures of the target compounds were confirmed with ¹H NMR, ¹³C NMR, MS and element analysis. Anti-tumor activities against HCT-116, MDA-MB435 and HL-60 cell lines in vitro were evaluated by SRB assay. The results indicated most of the target compounds exhibited potent anti-tumor activity. Compound 4a showed the most potent inhibitory activities against three cancer cell lines with the GI₅₀ values of 1.904-2.111 μmol·L^-1. Compounds 4g, 4l-4n exhibited more potent inhibitory activities against MDA-MB435 and HL-60 cell lines with the GI₅₀ values of 1.008-3.586 μmol·L^-1 and 1.993-3.778 μmol·L^-1, respectively. The structure-activity relationship of these compounds is discussed preliminarily.