Renal fibrosis is the common final pathway in many renal diseases regardless of the underlying etiology. Adipocyte enhancer-binding protein 1 (AEBP1) was reported to play a vital role in the development of organ fibrosis, but its role in renal fibrosis has not been reported. Thus, the aim of this study was to investigate the possible function of AEBP1 in renal fibrosis and the mechanism associated with the β-catenin signaling pathway. A total of 83 genes upregulated after unilateral ureteral obstruction (UUO) were screened from two Gene Expression Omnibus (GEO) datasets and subjected to Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Among them, AEBP1 was enriched in collagen binding and the regulation of collagen fibril organization and was confirmed to be upregulated in UUO kidneys and TGF-β1-induced cells. Knockdown of AEBP1 ameliorated renal fibrosis via reducing collagen accumulation, inhibiting epithelial–mesenchymal transition and fibroblast transformation, as evidenced by decreases in the expression of collagen I and III, Col1a1, Col3a1, fibronectin, Snail, α-SMA, as well as collagen-specific staining of kidney tissues, whereas the E-cadherin was increased. Besides, AEBP1 silencing inhibited the expression of β-catenin in nucleus and β-catenin downstream proteins (Axin2, Myc, and Ccnd1). Continuously active β-catenin–S33Y further restored the inhibitory effect of AEBP1 silencing on renal fibrosis. These findings indicate that knockdown of AEBP1 could potentially slow down renal fibrosis by blocking the β-catenin signaling pathway, highlighting the potential of AEBP1 as a therapeutic target for renal fibrosis.