Aim To investigate the effect of overexpression of heme oxygenase-1(HO-1) gene on endothelial cells injury induced by high D-glucose and free fatty acid. Methods With gene recombination and transfer techniques, human HO-1 gene was tranfected into human umbilical vein endothelial cells(ECV304). Expression of HO-1 gene was analyzed in ECV304 and transfected into endothelial cells by using semi-quantitative reverse transcription polymerase chain reaction. Transfected cells and ECV304 were cultured in media containing D-glucose (5.5 mmol/L, 20 mmol/L) and/or palmitic acid (125 μmmol/L, 250 μmmol/L, 375 μmmol/L). After 48 h, cell viability in each group was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the release rate of lactate dehydrogenase was measured by colorimetric assay. Lipid peroxidation in cells was tested as malondialdehyde content. Results The expression level of HO-1 mRNA was increased significantly in transfected cells. Treatment of ECV304 cells with 20 mmol/L D-glucose and different concentration palmitic acid caused a great decrease in cell viability, and an increase in malondialdehyde contents and lactate dehydrogenase release rate. Transfected cells showed higher viability, lower malondialdehyde contents and lactate dehydrogenase release rate compaired to those untransfected cells with the same treatment. The protection effect of HO-1 was abolished when HO-1 inhibitor zinc protoporphyrin-Ⅸ was used in transfected cells. Conclusions These findings suggest that the upregulation of heme oxygenase-1 gene expression was able to enhance endothelial cell resistance against injury induced by high D-glucose and free fatty acid, which may be related to it's activity as antioxidant.