〔Abstract〕 To explore the role and mechanism of 25-hydroxycholesterol (25-HC) in inflammatory bow- el disease (IBD) in mice . Methods All mice were divided into three groups : the control group was fed normally; the DSS model group was fed with 2. 5% dextran sulfate sodium (DSS) solution; the DSS + 25-HC experimental group was fed with 2. 5% DSS solution and he mice in the experimental group were intraperitoneally injected with 25-HC . The symptom changes of the mice were evaluated by assessing the disease activity index(DAI) , and the tis- sue changes were judged by histological scoring. The expression of interleukin-17 and its signaling pathways in the mice were detected by Western blot ,qRT-PCR , immunohistochemistry/fluorescence , and flow cytometry. Combined with the detection of tight junction proteins in the intestinal epithelium of the mice , the mechanism by which 25-HC affects IBD in mice was explored . Results In comparison to the DSS control group , The DSS + 25-HC experimen- tal group mice exhibited a reduction in body weight ( F = 30. 1 , P < 0. 000 1) , a shortened colon ( F = 63 . 8 , P < 0. 05) , and elevated DAI(F = 774. 5 , P < 0. 000 1) and histopathological scores(F = 141 . 5 , P < 0. 05) . Addition- ally , the expression of tight junction-associated proteins(ZO-2 , Occludin , JAM and Claudin-4) was found to be sig- nificantly reduced . The level of IL-17 significantly decreased , and its expression level was positively correlated with tight junction proteins . Conclusion 25-HC inhibited IL-17 production by colonic γδT cells through the RORγt pathway, aggravated mucosal injury , and promoted the development of DSS-induced acute colitis in mice .