The epithelial-to-mesenchymal transition (EMT) induced by chronic hypoxia is one of the critical causes of renal fibrosis. Previous work reported that the transcription factors Twist plays an important role in hypoxia-induced EMT and renal fibrosis. Recent evidence indicates that miR-214 was regulated by Twist in many fibrotic diseases, but their role in hypoxia-induced EMT and renal fibrosis remains unknown. Here, we found that hypoxia significantly upregulated the expression of miR-214-3p in HK-2 cells, unilateral ureteral obstruction (UUO) nephropathy and patients with chronic kidney disease. Knockdown of miR-214-3p reversed the EMT of renal tubular epithelial cells (TECs) and alleviated fibrosis in the UUO mouse in vivo, while the overexpression of miR-214-3p promoted EMT phenotype and expression of fibrotic factors in TECs under hypoxic condition. In addition, Twist was also observed increased gradually with the prolongation of hypoxia, and it positively correlated with the expression of miR-214-3p in HK-2 cells transfected with Twist-overexpression or Twist-siRNA plasmid. Moreover, miR-214-3p negatively regulated the expression of epithelial cadherin (E-cadherin) by binding the E-cadherin 3' UTR under hypoxic condition. Overall, hypoxia-induced activation of Twist/miR-214/E-cadherin axis is involved in the EMT of TECs, and anti-miR-214 may be an attractive strategy to ameliorate the progression of renal fibrosis.