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Background: <i>Tropheryma whipplei</i> (<i>TW</i>) can cause different pathologies, e.g., Whipple’s disease and transient gastroenteritis. The mechanism by which the bacteria pass the intestinal epithelial barrier, and the mechanism of <i>TW</i>-induced gastroenteritis are currently unknown. Methods: Using ex vivo disease models comprising human duodenal mucosa exposed to <i>TW</i> in Ussing chambers, various intestinal epithelial cell (IEC) cultures exposed to <i>TW</i> and a macrophage/IEC coculture model served to characterize endocytic uptake mechanisms and barrier function. Results: <i>TW</i> exposed ex vivo to human small intestinal mucosae is capable of autonomously entering IECs, thereby invading the mucosa. Using dominant-negative mutants, <i>TW</i> uptake was shown to be dynamin- and caveolin-dependent but independent of clathrin-mediated endocytosis. Complementary inhibitor experiments suggested a role for the activation of the Ras/Rac1 pathway and actin polymerization. <i>TW</i>-invaded IECs underwent apoptosis, thereby causing an epithelial barrier defect, and were subsequently subject to phagocytosis by macrophages. Conclusions: <i>TW</i> enters epithelia via an actin-, dynamin-, caveolin-, and Ras-Rac1-dependent endocytosis mechanism and consecutively causes IEC apoptosis primarily in IECs invaded by multiple <i>TW</i> bacteria. This results in a barrier leak. Moreover, we propose that <i>TW</i>-packed IECs can be subject to phagocytic uptake by macrophages, thereby opening a potential entry point of <i>TW</i> into intestinal macrophages.