Meprin metalloproteases are highly expressed at the luminal interface of the intestine and kidney and in certain leukocytes. Meprins cleave a variety of substrates in vitro including extracellular matrix proteins, adherens junction proteins and cytokines, and have been implicated in a number of inflammatory diseases. The linkage between results in vitro and pathogenesis however, has not been elucidated. The current study aimed to determine whether meprins are determinative factors in disrupting the barrier function of the epithelium. Active meprin A or meprin B applied to the Madin-Darby canine kidney (MDCK) cell monolayers increased permeability to fluorescein isothiocyanate (FITC)-dextran and disrupted immunostaining of the tight junction protein occludin but not claudin-4. Meprin A, but not meprin B, cleaved occludin in MDCK monolayers. Studies with recombinant occludin demonstrated meprin A cleaves the protein between Gly100 and Ser101 on the first extracellular loop. In vivo experiments demonstrated that meprin A infused into the mouse bladder increased the epithelium permeability to sodium fluorescein. Furthermore, monocytes from meprin knockout (KO) mice on the C57BL/6 background were less able to migrate through an MDCK monolayer than monocytes from wild-type (WT) counterparts. These results demonstrate the capability of meprin A to disrupt epithelial barriers and implicate occludin as one of the important targets of meprin A that may modulate inflammation.