Renal fibrosis is a common feature of chronic kidney disease, in which transforming growth factor-β1 (TGF-β1) plays an important role. Tripartite motif-containing 21 (TRIM21), an E3 ubiquitin ligase, has been studied for its role in acute kidney injury, but its role in renal fibrosis has not been reported. We analyzed public RNA-seq data of unilateral ureteral obstruction (UUO), ischemia-reperfusion injury (I/R), and aristolochic acid (AA)-induced renal fibrosis and found that TRIM21 expression was significantly elevated in fibrotic kidneys, which was verified by Western blot results corresponding to the mouse models. Similarly, TRIM21 expression was significantly elevated and negatively correlated with renal function in human fibrotic kidneys. We showed that TRIM21 knockout alleviated renal fibrosis in UUO mice. In vitro, TRIM21 knockout reduced TGF-β1-induced expression of mature TGF-β1 in HK-2 cells and primary renal tubular cells (PTECs), and this process was reversed by the autophagy inhibitor bafilomycin A1 (Baf-A1). Specifically, TRIM21 promoted K63-linked ubiquitination of p62, inhibited its oligomerization and thus its aggregation and segregation functions, and suppressed autophagic degradation of TGF-β1. Meanwhile, in the UUO mouse model, TRIM21 knockout promoted autophagy levels, and reduced the protein levels of mature TGF-β1 and the phosphorylation levels of SMAD2/3. In conclusion, our study demonstrates that TRIM21 knockdown alleviates renal fibrosis by promoting autophagic degradation of mature TGF-β1 and provides an insight into TRIM21 as a potential therapeutic target for the treatment of kidney fibrosis.