Colitis, an inflammatory disease of the digestive tract, is increasing in incidence and prevalence. Intestinal inflammation can occur as a consequence of dysfunctions in sphingolipid metabolism. In this study we used ceramide synthase 6 (CerS6) deficient mice, which have a reduced ability to generate long chain C16-ceramide, to investigate the role of this enzyme in dextran sodium salt (DSS)-induced colitis. While CerS6-deficient mice are protected from T cell mediated colitis, in the T cell independent DSS model lack of CerS6 resulted in a more rapid onset of disease symptoms. CerS6-deficient mice maintained low levels of C16-ceramide after DSS treatment, but the inflammatory lipid sphingosine-1-phosphate was significantly increased in colon tissue. In the absence of CerS6, DSS induced more severe pathology in the colon including enhanced neutrophil infiltration. In vivo analysis of myeloperoxidase activity, an enzyme released from neutrophils, was approximately 2.5-fold higher in CerS6-deficient mice compared to wild type. Differences in intestinal permeability did not account for the increase in neutrophils. Our study suggests that lack of CerS6 expression differentially impacts the development of colitis, depending on the model used.