〔Abstract〕 Objective To investigate the protective effect of protein disulfide isomerase A3(PDIA3) on ulcerative colitis(UC) mice and its possible mechanism. Methods Sixty C57 BL/6 mice were randomly divided into a normal Control group(Control), Model group(Model), no-load lentivirus group(Lv-NC) and a PDIA3 overexpressed lentivirus group(Lv-PDIA3), with 15 mice in each group. The mouse model of UC was established by drinking 3% dextran sulfate sodium(DSS) solution for 7 consecutive days. After successful modeling, lentivirus intervention was started. After 7 days of lentivirus intervention, the mice in each group were evaluated by disease activity index(DAI) score. The histopathological changes of colon were observed by HE staining. The inflammatory cytokines of interleukin 1β(IL-1β),interleukin 6(IL-6),tumor necrosis factor α(TNF-α), and the intestinal mucosal barrier permeability markers of D-lactic acid(D-Lac), diamine oxidase(DAO) levels in serum were detected by ELISA, PDIA3 mRNA levels in colon tissues were detected by qRT-PCR. The expression levels of PDIA3, ZO-1, Occludin, Claudin-1, IκBα, p-NF-κB p65(Ser536) and NF-κB p65 in colonic tissue were detected by Western blot. Results Compared with the Control group, the colon tissue of the Model group was severely damaged. DAI score, the levels of IL-1β, IL-6, TNF-α, D-Lac, DAO in serum, and the expression levels of IκBα and p-NF-κB p65(Ser536) in colon tissues were increased(P<0.01). The proteins expression levels of ZO-1, Occludin and Claudin-1 in colon tissues were reduced(P<0.01). Compared with the Model group, the damage degree of colonic tissue in Lv-PDIA3 group was reduced. DAI score, the levels of IL-1β, IL-6, TNF-α, D-Lac, DAO in serum, and the expression levels of IκBα and p-NF-κB p65(Ser536) in colon tissues were decreased(P<0.01). The proteins expression levels of ZO-1, Occludin and Claudin-1 in colon tissues were increased(P<0.01). However, there was no significant difference in Lv-NC group(P>0.05). Conclusion Overexpression of PDIA3 improved the impaired intestinal mucosal barrier function in UC mice, which may be related to inhibition of NF-κB signaling pathway activation.