Renal fibrosis is a progressive pathological change characterized by tubular cell apoptosis, tubulointerstitial fibroblast proliferation, and excessive deposition of extracellular matrix (ECM). miR-21 has been implicated in transforming growth factor- (TGF-)-stimulated tissue fibrosis. Recent studies showed that sphingosine kinase/sphingosine-1-phosphate (SphK/S1P) are also critical for TGF--stimulated tissue fibrosis; however, it is not clear whether SphK/S1P interacts with miR-21 or not. In this study, we hypothesized that SphK/S1P signaling is linked to upregulation of miR-21 by TGF-. To verify this hypothesis, we first determined that miR-21 was highly expressed in renal tubular epithelial cells (TECs) stimulated with TGF- by using qRT-PCR and Northern blotting. Simultaneously, inhibition of miR-21, mediated by the corresponding antimir, markedly decreased the expression and deposition of type I collagen, fibronectin (Fn), cysteine-rich protein 61 (CCN1), -smooth muscle actin, and fibroblast-specific protein1 in TGF--treated TECs. ELISA and qRT-PCR were used to measure the S1P and SphK1 levels in TECs. S1P production was induced by TGF- through activation of SphK1. Furthermore, it was observed that TGF--stimulated upregulation of miR-21 was abolished by SphK1 siRNA and was restored by the addition of exogenous S1P. Blocking S1PR(2) also inhibited upregulation of miR-21. Additionally, miR-21 overexpression attenuated the repression of TGF--stimulated ECM deposition and epithelial-mesenchymal transition by SphK1 and S1PR(2) siRNA. In summary, our study demonstrates a link between SphK1/S1P and TGF--induced miR-21 in renal TECs and may represent a novel therapeutic target in renal fibrosis.