Evidence from inflammatory bowel disease (IBD) patients and animal models suggests that inflammation alters blood flow to the mucosa, which precipitates mucosal barrier dysfunction. Impaired purinergic sympathetic regulation of submucosal arterioles, the resistance vessels of the splanchnic vasculature, is one of the defects identified during IBD and in mouse models of IBD. We hypothesized that this may be a consequence of upregulated catabolism of ATP during colitis. In vivo and in vitro videomicroscopy techniques were employed to measure the effects of purinergic agonists and inhibitors of CD39, an enzyme responsible for extracellular ATP catabolism, on the diameter of colonic submucosal arterioles from control mice and mice with dextran sulphate sodium (DSS; 5% w/v) colitis. We examined the degradation of ATP using a luciferase-based ATP assay and utilized real time-PCR, Western blotting and immunohistochemistry to examine the expression and localization of CD39 during colitis. Arterioles from mice with DSS colitis did not constrict in response to ATP (10 µM) but did constrict in the presence of its non-hydrolyzable analogue, -methylene ATP (1 µM). -methylene ADP (100 µM), an inhibitor of CD39, restored ATP-induced vasoconstriction in arterioles from DSS colons. CD39 protein and mRNA expression was markedly increased in colitis. Immunohistochemical analysis demonstrated that in addition to vascular CD39, F4/80 immunoreactive macrophages accounted for a large proportion of submucosal CD39 staining in colitis. These data implicate upregulation of CD39 in impaired sympathetic regulation of gastrointestinal blood flow during colitis.