Background & Aims: The diarrheagenic pathogen, enteropathogenic Escherichia coli (EPEC), uses a type III secretion system to deliver effector molecules into intestinal epithelial cells (IECs). While exploring the basis for the lateral membrane separation of EPEC-infected IECs, we observed infection-induced loss of the desmosomal cadherin desmoglein-2 (DSG2). We sought to identify the molecule(s) involved in, and delineate the mechanisms and consequences of, EPEC-induced DSG2 loss. Methods: DSG2 abundance and localization was monitored via immunoblotting and immunofluorescence, respectively. Junctional perturbations were visualized by electron microscopy, and cell-cell adhesion was assessed using dispase assays. EspH alanine-scan mutants as well as pharmacologic agents were used to evaluate impacts on desmosomal alterations. EPEC-mediated DSG2 loss, and its impact on bacterial colonization in vivo, was assessed using a murine model. Results: The secreted virulence protein EspH mediates EPEC-induced DSG2 degradation, and contributes to desmosomal perturbation, loss of cell junction integrity, and barrier disruption in infected IECs. EspH sequesters Rho guanine nucleotide exchange factors and inhibits Rho guanosine triphosphatase signaling; EspH mutants impaired for Rho guanine nucleotide exchange factor interaction failed to inhibit RhoA or deplete DSG2. Cytotoxic necrotizing factor 1, which locks Rho guanosine triphosphatase in the active state, jasplakinolide, a molecule that promotes actin polymerization, and the lysosomal inhibitor bafilomycin A, respectively, rescued infected cells from EPEC-induced DSG2 loss. Wild-type EPEC, but not an espH-deficient strain, colonizes mouse intestines robustly, widens paracellular junctions, and induces DSG2 re-localization in vivo. Conclusions: Our studies define the mechanism and consequences of EPEC-induced desmosomal alterations in IECs. These perturbations contribute to the colonization and virulence of EPEC, and likely related pathogens.