Aim Glutathione-S-transferases (GST), enzymes with antioxidant activity, have been shown to play a protective role against oxidative stress for many cell types including human vascular smooth muscle cells, but their role in nitroglycerin (NTG) tolerance specifically has not been demonstrated. In this study, we tested the role of hGSTA4-transfection in conferring resistance to the development of tolerance on human fetal aortic smooth muscle cells (FASMC) treated with NTG. Methods A stable transfection of FASMC with cDNA of hGSTA4-4 was established. Western blot analysis, immunocytochemistry assay, MTT cytotoxicity assay and NO assay were used. Results FASMC overexpressing with the hGSTA4-4 were significantly more resistance to cytotoxic injury by NTG, assessed at 24 hours (P<0.05). NO release was higher in hGSTA4-transfected cells during NTG nontolerance and tolerance treatment compared with those in wild-type and vector-transfected FASMC (P<0.01). Conclusions This study demonstrates that hGSTA4-4 can protect smooth muscle cells from the cytotoxicity of NTG and may have a role in smooth muscle cell tolerance to NTG.