NOX1, an NADPH oxidase predominantly expressed in colon epithelial cells, has emerged as a prime regulator of mucosal immunity and homeostasis through its ability to coordinate several important epithelial cell functions. A critical question still unanswered is how the production of reactive oxygen species (ROS) by NOX1 is controlled in vivo to allow the beneficial effects while avoiding oxidative damage and diseases from these inherently toxic molecules. Here, we report that the ubiquitous protein kinase CK2 limits NOX1 activity in colonic epithelial cells and lessens experimental colitis. Using several complementary approaches, we identified CK2 as a major partner of the NOX1 organizer subunit, NOXO1, in colon epithelial cells under inflammatory conditions. CK2 phosphorylated NOXO1 on several sites, as shown by mass spectrometry, and the highly selective CK2 inhibitor, CX-4945, increased ROS generation by NOX1 in human colon epithelial cells and organoids. Strikingly, CK2 activity dropped in TNBS-induced acute colitis while NOX1 expression increased, and CX-4945 exacerbated colitis inflammation as shown by increased CXCL1, ROS, lipid peroxidation and colon damage. Our data suggest that CK2 acts as a molecular brake to control NOX1 activation and that loss of CK2 activity during acute colitis may result into excessive ROS production, contributing to pathogenesis. Strategies to activate CK2 could be an effective novel therapeutic approach in inflammatory bowel diseases.