Orphan G protein-coupled receptor GPR35, which is activated by lysophosphatidic acid and kynurenic acid, is highly expressed in gastrointestinal tracts. Clinical findings indicate that this receptor has been implicated in the onset of inflammatory bowel disease (IBD), but its role in physiological and pathological processes in the colon remains undefined. The present study investigated the role of GPR35 in the pathogenesis of exmerimentally-induced colitis in mice.GPR35-deficient (GPR35KO) mice were generated by CRISPR-Cas9-mediated genome editing on C57BL/6 background. Experimental colitis was induced in GPR35KO and wild-type (WT) mice by the treatment with dextran sulfate sodium (DSS) for 7 days. Lodoxamide, which has a GPR35 activating effect, was administered i.p. once daily for 7 days. DSS treatment produced body weight loss with diarrhea and blood feces, and severe colitis characterized by shortening colon length and histological injury 7 days later. The severity of colitis with systemic symptoms was significantly augmented in GPR35KO mice compared with WT mice. In contrast, daily administration of lodoxamide significantly reduced the severity of DSS-induced colitis in WT mice. However, the protective effect of lodoxamide was not observed in GPR35KO mice. These findings suggest that GPR35 plays an anti-inflammatory role in DSS-induced colitis. Thus, GPR35 may be a promising target for treatment and prevention of inflammatory bowel disease.