Nuclear receptor 4A1 (NR4A1) is a gene that increases the likelihood of chronic kidney disease (CKD) and contributes to its development. Previous research has shown that the SAM pointed domain containing Ets transformation-specific transcription factor (SPDEF) can activate NR4A1, but its mechanism of action in renal fibrosis is not yet clear. In this study, we used adenovirus to create a mouse kidney model with a specific knockdown of NR4A1 gene. Our results showed that the knockdown of NR4A1 can accelerate unilateral ureteral obstruction (UUO)-induced renal fibrosis in mice, and overexpression of NR4A1 can significantly reduce transforming growth factor-β1-induced (TGF-β1) fibrosis in HK-2 cells. Additionally, we found that overexpression of SPDEF can improve UUO-induced renal fibrosis in mice and TGF-β1-induced fibrosis in HK-2 by transcriptionally activating NR4A1. These findings suggest that SPDEF can activate NR4A1 transcriptionally and improve renal fibrosis.© 2024. The Author(s).