Epithelial-mesenchymal transition (EMT) contributes to the progression of renal tubulointerstitial fibrosis. The N-methyl-d-aspartate receptor (NMDAR), which is present in proximal tubular epithelium, is a glutamate receptor that acts as a calcium channel. Activation of NMDAR induces actin rearrangement in cells of the central nervous system, but whether it helps maintain the epithelial phenotype of the proximal tubule is unknown. Here, knockdown of NMDAR1 in a proximal tubule cell line (HK-2) induced changes in cell morphology, reduced E-cadherin expression, and increased alpha-SMA expression. Induction of EMT with TGF-beta1 led to downregulation of both E-cadherin and membrane-associated beta-catenin, reorganization of F-actin, expression of mesenchymal markers de novo, upregulation of Snail1, and increased cell migration; co-treatment with NMDA attenuated all of these changes. Furthermore, NMDA reduced TGF-beta1-induced phosphorylation of Erk1/2 and Akt and the activation of Ras, suggesting that NMDA antagonizes TGF-beta1-induced EMT by inhibiting the Ras-MEK pathway. In the unilateral ureteral obstruction model, treatment with NMDA blunted obstruction-induced upregulation of alpha-SMA, FSP1, and collagen I and downregulation of E-cadherin. Taken together, these results suggest that NMDAR plays a critical role in preserving the normal epithelial phenotype and modulating tubular EMT.