Vibrio cholerae ‐derived zonula occludins toxin (Zot) is a multifunctional protein that reversibly disassembles intestinal tight junctions (tjs). Zot structure‐function analysis has mapped this activity to aa 288–293, named AT1002. AT1002 reduced transepithelial electrical resistance across rat small intestine, ex vivo, as did Zot and its processed mature form, ΔG. AT1002 increased in vivo permeability to sugar tracers, whereas scrambled control peptides did not. Binding and barrier assays in proteinase activated receptor (PAR) ₂ ‐expressing and PAR ₂ ‐null cells established AT1002 activity to be PAR ₂ dependent. Coincident with the increased intestinal permeability, confocal microscopy of AT1002‐exposed rat intestinal IEC6 cells revealed displacement of ZO‐1 and occludin from intercellular boundaries. In coimmunoprecipitation assays, AT1002 decreased ZO‐1‐occludin and ZO‐1‐claudin 1 interactions coincident with PKCα‐dependent ZO‐1 serine/threonine phosphorylation. Further, AT1002 increased serine phosphorylation of myosin 1C and, at the same time, transiently diminished its association with ZO‐1. The COOH‐terminal domain of ZO‐1 was required for its association with myosin 1C. These data indicate that the NH ₂ ‐terminal portion of active Zot contains a PAR ₂ ‐activating motif, FCIGRL, that increases PKCα‐dependent ZO‐1 and myosin 1C serine/threonine phosphorylation. These modifications provoke selective disengagement of ZO‐1 from its binding partners, occludin, claudin 1, and myosin 1C, coincident with opening of tjs.—Goldblum, S. E., Rai, U., Tripathi, A., Thakar, M., De Leo, L., Di Toro, N., Not, T., Ramachandran, R., Puche, A. C., Hollenberg, M. D., Fasano, A. The active Zot domain (aa 288–293) increases ZO‐1 and myosin 1C serine/ threonine phosphorylation, alters interaction between ZO‐1 and its binding partners, and induces tight junction disassembly through proteinase activated receptor 2 activation. FASEB J. 25, 144–158 (2011). www.fasebj.org