Renal fibrosis is the final pathological outcome of chronic kidney disease and is characterized by an excessive accumulation of extracellular matrix (ECM), leading to tissue dysfunction and, ultimately, organ failure. In this study, we investigated the mechanism by which elevated intracellular sphingosine levels in sphingosine kinase 2-deficient (Sphk2) fibroblasts and mice attenuate profibrotic TGF-β signaling. Previously, we demonstrated that higher sphingosine levels suppress the expression of profibrotic factors in TGF-β-treated fibroblasts and a unilateral ureteral obstruction (UUO) mouse model by upregulating SMAD7, an inhibitor of TGF-β/SMAD signaling. Here, we identified miR-21 as a key post-transcriptional regulator of SMAD7 and show that protein kinase C (PKC) inhibition-via sphingosine supplementation, SPHK2 depletion, or pharmacological PKC inhibitors-reduces miR-21 expression. Mechanistically, this occurs through PKC-dependent inhibition of AP1-driven miR-21 transcription. Analysis of human diabetic kidney tissue revealed a significant upregulation of miR-21, contributing to ECM accumulation by suppressing SMAD7 protein expression, corroborating the critical role of miR-21 in renal fibrosis. These findings suggest that targeting the sphingosine-PKC-miR-21-SMAD7 axis may offer a novel therapeutic approach for renal fibrosis.