Transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) contributes to the pathophysiological development of kidney fibrosis. Although it was reported that TGF-β1 enhances β₁ integrin levels in NMuMG cells, the detailed molecular mechanisms underlying TGF-β1-induced β₁ integrin gene expression and the role of β₁ integrin during EMT in the renal system are still unclear. In this study, we examined the role of β₁ integrin in TGF-β1-induced EMT both in vitro and in vivo. TGF-β1-induced augmentation of β₁ integrin expression was required for EMT in several epithelial cell lines, and knockdown of Smad3 inhibited TGF-β1-induced augmentation of β₁ integrin. TGF-β1 triggered β₁ integrin gene promoter activity as assessed by luciferase activity assay. Both knockdown of Smad3 and mutation of the Smad-binding element to block binding to the β₁ integrin promoter markedly reduced TGF-β1-induced β₁ integrin promoter activity. Chromatin immunoprecipitation assay showed that TGF-β1 enhanced Smad3 binding to the β₁ integrin promoter. Furthermore, induction of unilateral ureteral obstruction triggered increases of β₁ integrin in both renal epithelial and interstitial cells. In human kidney with chronic tubulointerstitial fibrosis, we also found a concomitant increase of β₁ integrin and α-smooth muscle actin in tubule epithelia. Blockade of β₁ integrin signaling dampened the progression of fibrosis. Taken together, β₁ integrin mediates EMT and subsequent tubulointerstitutial fibrosis, suggesting that inhibition of β₁ integrin is a possible therapeutic target for prevention of renal fibrosis.