The host metalloprotease meprin β is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease. Display omitted •Meprin β activation and shedding are mutually exclusive events•ADAM-mediated pro-meprin β shedding is required for proper mucus integrity•Pathogenic secreted cysteine protease RgpB activates host metalloprotease meprin β•Activation of membrane-bound meprin β prevents its shedding and mucus detachment ADAM-mediated meprin β shedding is required for mucus detachment, regulating intestinal integrity. This work by Wichert et al. demonstrates that meprin β is exclusively shed in its pro-form. Activation of meprin β by the serine protease MT-2 or the bacterial virulence factor RgpB abrogates its shedding, resulting in a disturbed mucus barrier.