Aim To investigate whether N-acetylcysteine (NAC) can protect endothelial cells from high glucose induced injury by inhibiting JAK2 /STAT3 signaling pathway. Methods Human umbilical vein endothelial cells (HUVEC) were pretreated with different concentrations of NAC in vitro, and the optimal concentration of NAC was selected to reduce the cytotoxicity of HUVEC induced by high glucose. HUVEC were pretreated with the best concentration of NAC, afterwards ,the survival rate of HUVEC was measured by CCK-8, the morphological changes of endothelial cell apoptosis were detected by Hoechst33258 nuclear staining, the protein expression of JAK2/STAT3 signaling pathway was detected by Western blot, and the level of intracellular reactive oxygen species was detected by DCFH-DA, the levels of intercellular cell adhesion molecule-1 (ICAM-1), nuclear factor-κB (NF-κB), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6 and IL-8 were detected by ELISA, and the changes of mitochondrial membrane potential were detected by fluorescence probe JC-1. Results NAC pretreatment of HUVEC for 30 min could significantly reduce the injury induced by high glucose, increase the cell survival rate, reduce the number of apoptosis, decrease the expression of cleaved Caspase-3, decrease the accumulation of intracellular reactive oxygen species and the loss of mitochondrial membrane potential (P＜0.01). NAC can inhibit the up-regulation of p-JAK2 and p-STAT3 expression induced by high glucose (P＜0.01), inhibit the inflammatory response induced by high glucose, and decrease the levels of inflammatory factors such as ICAM-1, NF-κB, TNF-α, IL-1β, IL-6 and IL-8 (P＜0.01). Conclusion NAC can protect HUVEC from high glucose induced injury by inhibiting JAK2/STAT3 signaling pathway.