Inflammatory bowel disease (IBD) has emerged as a public health challenge with high incidence, recurrence rates and low cure rate. Moreover, sustained inflammation increases the risk of colorectal cancer. The occurrence and progression of IBD are closely related to the genetic mutation. Previous genome-wide association studies (GWAS) analysis demonstrated that the susceptibility loci rs4676410, rs3749171, and rs3749172 in theGPR35 gene locus increase the risk of IBD, but no direct evidence on the function ofGPR35 in IBD progression has been shown. To investigate the role ofGPR35 in IBD, CRISPR/Cas9 technology was employed to construct aGpr35 knockout mouse strain. TheGpr35 mice exhibited lower susceptibility to dextran sodium sulfate-induced IBD model than the wildtype group with a significant reduction in body weight loss, DAI score, intestinal epithelial injury, and macrophage cell infiltration. To explore how the IBD susceptibility loci rs3749171 and rs3749172 regulate GPR35 activity, two mutant forms of GPR35 (T108M and S294R) were constructed. By analyzing the activity of GPR35 downstream signaling pathway, the two mutation forms of GPR35 exhibited higher receptor activity to Zaprinast than the wildtype GPR35. Finally, the Western blotting analysis found an elevated phosphorylation level of ERK1/2 inGpr35 colon epithelial after DSS treatment, demonstrating that the loss function ofGpr35 alleviates the IBD syndrome by activating the ERK1/2 signaling pathway. In summary, the IBD susceptibility loci rs3749171 and rs3749172 may promote the disease progression by activating GPR35 activity, providing a potential drug target for the treatment of inflammatory bowel disease.