Inflammatory diseases of the digestive tract, including inflammatory bowel disease (IBD), 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 IBD 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 CKB/CKMit (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 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. We identified basal stabilization of the transcription factor hypoxia-inducible factor (HIF) in CKdKO splenocytes and pharmacological stabilization of HIF 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.