BACKGROUND: Intestinal fibrosis, a complex complication of colitis, is characterized by excessive extracellular matrix (ECM) deposition. Estrogen receptor (ER) beta may play a role in regulating this process. METHODS: Intestinal tissue samples from stenotic and nonstenotic regions were collected from Crohn's disease (CD) patients. RNA sequencing was conducted on a mouse model to identify differentially expressed mRNAs. Histological, immunohistochemical, and semiquantitative Western blotting analyses were employed to assess ECM deposition and fibrosis. The roles of relevant pathways in fibroblast transdifferentiation, activity, and migration were examined. RESULTS: Estrogen receptor beta expression was found to be downregulated in the stenotic intestinal tissue of CD patients. Histological fibrosis score, collagen deposition, and profibrotic molecules in the colon of an intestinal fibrosis mouse model were significantly decreased after activation of ERbeta. In vitro, ERbeta activation alleviated transforming growth factor (TGF)-beta-induced fibroblast activation and migration, as evidenced by the inhibition of col1alpha1, fibronectin, alpha-smooth muscle actin (alpha-SMA), collagen I, and N-cadherin expression. RNA sequencing showed that ERbeta activation affected the expression of genes involved in ECM homeostasis and tissue remodeling. Enrichment analysis of differentially expressed genes highlighted that the downregulated genes were enriched in ECM-receptor interaction, TGF-beta signaling, and Toll-like receptor (TLR) signaling. Western blotting confirmed the involvement of TGF-beta/Smad and TLR4/MyD88/NF-kappaB signaling pathways in modulating fibrosis both in vivo and in vitro. The promoter activity of TGF-beta1 and TLR4 could be suppressed by ERbeta transcription factor. CONCLUSION: Estrogen receptor beta may regulate intestinal fibrosis through modulation of the TGF-beta/Smad and TLR4/MyD88/NF-kappaB signaling pathways. Targeting ERbeta activation could be a promising therapeutic strategy for treating intestinal fibrosis.