Inflammatory diseases of the digestive tract, including inflammatory bowel disease, cause metabolic stress within mucosal tissue. Creatine is a key energetic regulator. We previously reported a loss of creatine kinases (CKs) and the creatine transporter expression in inflammatory bowel disease patient intestinal biopsy samples and that creatine supplementation was protective in a dextran sulfate sodium (DSS) colitis mouse model. In the present studies, we evaluated the role of CK loss in active inflammation using the DSS colitis model. Mice lacking expression of CK brain type/CK mitochondrial form (CKdKO) showed increased susceptibility to DSS colitis (weight loss, disease activity, permeability, colon length, and histology). In a broad cytokine profiling, CKdKO mice expressed near absent interferon gamma (IFN-γ) levels. We identified losses in IFN-γ production from CD4 and CD8 T cells isolated from CKdKO mice. Addback of IFN-γ during DSS treatment resulted in partial protection for CKdKO mice. Extensions of these studies identified basal stabilization of the transcription factor hypoxia-inducible factor in CKdKO splenocytes and pharmacological stabilization of hypoxia-inducible factor resulted in reduced IFN-γ production by control splenocytes. Thus, the loss of IFN-γ production by CD4 and CD8 T cells in CKdKO mice resulted in increased colitis susceptibility and indicates that CK is protective in active mucosal inflammation.