Orphan G protein coupled receptor GPR35 is highly expressed in gastrointestinal tracts. We previously reported that GPR35 plays a protective role in the pathogenesis of colitis via promoting tissue repair and healing, but the detail mechanism is not fully understood. The present study investigated the protective role of GPR35 in the pathogenesis of dextran sulfate sodium (DSS)-induced colitis, especially in relation to epithelial barrier functions and inflammatory responses. Colitis was induced in male GPR35-deficient (GPR35KO) and wild-type (WT) mice by DSS treatment for 7 days. For intestinal epithelial barrier functions, mucus secretion and tight/adherence junction protein expression were examined. For inflammatory responses, cytokine expression was examined in bone marrow-derived macrophages (BMDM). DSS treatment produced severe colitis accompanied by body weight loss and diarrhea/bloody stool, but the severity was significantly aggravated in GPR35KO mice compared with WT mice. There is no difference in epithelial barrier functions between both mice. In contrast, LPS-induced upregulation of cytokine expression significantly enhanced in BMDM obtained from GPR35KO mice compared with WT mice. Further, lysophosphatidic acid, a GPR35 agonist, suppressed LPS-induced cytokine expression in BM from WT mice but not GPR35KO mice. These results suggest that GPR35 plays a protective role in the pathogenesis of DSS-induced colitis. This response may be mediated by attenuation of inflammatory responses but not regulating epithelial barrier functions.