Orphan G protein-coupled receptor GPR35, which can be activated by lysophosphatidic acid and kynurenic acid, is highly expressed in gastrointestinal tracts. Previous studies demonstrate the implication of GPR35 in the pathogenesis of inflammatory bowel disease but this role remains undefined. The present study investigated the pathogenic role of GPR35 in murine ileitis and colitis models. GPR35-deficient (GPR35KO) mice were generated by CRISPR-Cas9-mediated genome editing on C57BL/6 background. Colitis and ileitis were induced in GPR35KO and its wild-type (WT) mice by 7-days treatment with dextran sulfate sodium (DSS) and single injection of indomethacin, respectively. DSS-treatment produced body weight loss with diarrhea and blood feces, and caused 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. Indomethacin injection produced intestinal injury 48 h later and the severity was also more higher in GPR35KO mice than WT mice. These findings suggest that GPR35 plays an anti-inflammatory role in DSS-induced colitis and indomethacin-induced ileitis in mice. Thus, GPR35 may be a promising target for treatment of inflammatory bowel disease.