Objective To investigate the protective effect of n-acetylcysteine (NAC) on injury of human renal tubular epithelial cells (HK-2 cells) induced by methylglyoxal and its underlying mechanism. Methods HK-2 cells were divided into six group: control group (no treatment), injury group (induced by methylglyoxal) and NAC groups (treated with different concentrations of NAC, 1, 2, 4, 8 mmol/L). Cell viability was measured by CCK- 8. Morphological changes of apoptotic cell nucleus were observed by DAPI staining. Mitochondrial membrane potential (MMP) was detected by rhodamine 123 staining and flow cytometry. Expression of Bax, Bcl-2, Caspase-3 and p-ERK1/2 were identified by western blot. Results Compared with control group, cell viability, MMP and the expression of Bcl-2 of HK-2 cells were significantly reduced by methylglyoxal while the number of apoptotic cells and the protein level of Bax, Cleaved Caspase-3 and p-ERK1/2 were dramatically increased. However, all of the above changes were reversed by NAC intervention. Conclusions NAC exerts protective effect on methylglyoxal induced injury of HK-2 cells. Potential mechanism may be associated with the anti-apoptotic effect and inhibition of activation of ERK1/2 cell signaling.