Objective : 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 γδ T cells through the RORγt pathway，aggravated mucosal injury，and promoted the development of DSS-induced acute colitis in mice．