5-androstenediol (ADIOL) functions as a selective estrogen receptor β (ERβ) ligand with a protective effect against many diseases. So, we conducted a novel insight into its role in acetic acid (AA)-induced colitis and investigated its effect on TLR4-Mediated PI3K/Akt and NF-κB Pathways and the potential role of ERβ as contributing mechanisms. Methods: Rats were randomized into 5 Groups; Control, Colitis, Colitis + mesalazine (MLZ), Colitis + ADIOL, and Colitis + ADIOL + PHTPP (ER-β antagonist). The colitis was induced through a rectal enema of acetic acid (AA) on the 8th day. At the end of treatment, colons were collected for macroscopic assessment. Tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), nuclear factor kappa b (NF-κB), toll-like receptor (TLR4), and phosphorylated Protein kinase B (pAKT) were measured. Besides, Gene expression of interleukin-1beta (IL-1β), metalloproteases 9 (Mmp9), inositol 3 phosphate kinase (PI3K), Neutrophil gelatinase-associated lipocalin (NGAL), ERβ and NLRP6 were assessed. Histopathological and immunohistochemical studies were also investigated. Results: Compared to the untreated AA group, the disease activity index (DAI) and macroscopic assessment indicators significantly decreased with ADIOL injections. Indeed, ADIOL significantly decreased colonic tissue levels of MDA, TLR4, pAKT, and NF-κB immunostainig while increased SOD activity and β catenin immunostainig. ADIOL mitigated the high genetic expressions of IL1β, NGAL, MMP9, and PI3K while increased ERβ and NLRP6 gene expression. Also, the pathological changes detected in AA groups were markedly ameliorated with ADIOL. The specific ERβ antagonist, PHTPP, largely diminished these protective effects of ADIOL. Conclusion: ADIOL could be beneficial against AA-induced colitis mostly through activating ERβ.