Compared with prostaglandin E₂, which has an established role in cancer, the role of the COX metabolite prostaglandin D₂ (PGD₂) in chronic inflammation leading to tumorigenesis is uncertain. In this study, we investigated the role of PGD₂ in colitis and colitis-associated colon cancer (CAC) using genetically modified mice and an established model of inflammatory colon carcinogenesis. Systemic genetic deficiency in hematopoietic PGD synthase (H-PGDS) aggravated colitis and accelerated tumor formation in a manner associated with increased TNFα expression. Treatment with a TNFα receptor antagonist attenuated colitis regardless of genotype. Histologic analysis revealed that infiltrated mast cells strongly expressed H-PGDS in inflamed colons. Mast cell–specific H-PGDS deficiency also aggravated colitis and accelerated CAC. In contrast, treatment with a PGD₂ receptor agonist inhibited colitis and CAC. Together, our results identified mast cell–derived PGD₂ as an inhibitor of colitis and CAC, with implications for its potential use in preventing or treating colon cancer. Cancer Res; 74(11); 3011–9. ©2014 AACR.