Background: Studies have shown that endothelial-to-mesenchymal transition (EndMT) could contribute to the progression of diabetic nephropathy, diabetic renal fibrosis, and cardiac fibrosis. The aim of this study was to investigate the influence of high glucose and related mechanism of MAPK inhibitor or specific antioxidant on the EndMT. Methods: In vitro human umbilical vein endothelial cells (HUVEC) were cultured with 11 mM, 30 mM, 60 mM and 120 mM glucose for 0, 24, 48, 72 and 168 h. Endothelial cell morphology was observed with microscope, and RT-PCR was used to detect mRNA expression of endothelial markers VE-cadherin and CD31, mesenchymal markers alpha-SMA and collagen I, and transforming growth factor TGF-beta 1. Immunofluorescence staining was performed to detect the expression of CD31 and alpha-SMA. The concentration of TGF-beta 1 in the supernatant was detected by ELISA. ERK1/2 phosphorylation level was detected by Western blot analysis. Results: High glucose induced EndMT and increased the TGF-beta 1 level in HUVEC cells. Cells in high glucose for 7 days showed a significant decrease in mRNA expression of CD31 and VE-cadherin, and a significant increase in that of a-SMA and collagen I, while lost CD31 staining and acquired alpha-SMA staining. ERK signaling pathway blocker PD98059 significantly attenuated the high glucose-induced increase in the ERK1/2 phosphorylation level. PD98059 and NAC both inhibited high glucose-induced TGF-beta 1 expression and attenuated EndMT marker protein synthesis. Conclusion: High glucose could induce HUVEC cells to undergo EndMT. NAC and ERK signaling pathway may play important role in the regulation of the TGF-beta 1 biosynthesis during high glucose-induced EndMT.