Background/Aims: Renal tubulointerstitial fibrosis is the most common pathway of progressive kidney injury, leading to end-stage renal disease. At present, no effective prophylactic treatment method is available. This study investigated the anti-fibrotic effects of total flavonoids (TFs) extracted from leaves of Carya Cathayensis in vivo and in vitro, and explored the underlying mechanisms. Methods: Anti-fibrotic effects of TFs were measured using a mouse model of unilateral ureteral obstruction (UUO) and in transforming growth factor-β1 (TGF-β1)-treated mouse tubular epithelial cells (mTECs). mRNA expression and protein levels of Collagen I, Collagen III, and α-smooth muscle actin (α-SMA) were also tested by real-time reverse transcription PCR and western blot analysis. To elucidate the underlying mechanisms, expression of miR-21 was examined in mTECs treated with TFs using miR-21 mimics transfected into mTECs before TGF-β1 and TFs treatment. Regulation of mothers against decapentaplegic homolog (Smad) signaling by miR-21 was subsequently validated via overexpression and deletion of miR-21 followed by a luciferase assay. Results: TFs treatment attenuated renal fibrosis, and inhibited expression of collagens and α-SMA in the kidneys of mice subjected to UUO. In vitro, the TFs significantly decreased expression of fibrotic markers in TGF-β1-treated mTECs. Moreover, TFs reduced miR-21 expression in a time- and dose-dependent manner in mTECs, increased expression of Smad7, and decreased phosphorylation of Smad3. Treatment with miR-21 mimics abolished the anti-fibrotic effects of the TFs on the TGF-β1-treated mTECs. In addition, genetic deletion of miR-21 upregulated expression of Smad7 and suppressed phosphorylation of Smad3, attenuating renal fibrosis in mice. Bioinformatics predictions revealed the potential binding site of miR-21 in the 3′-untranslated region of Smad7, and this was further confirmed by the luciferase assay. Conclusion: TFs ameliorate renal fibrosis via a miR-21/Smad7 signaling pathway, indicating a potential therapy for the prevention of renal fibrosis.