Objective: MVID is a congenital enteropathy characterized by loss of microvilli and formation of microvillus inclusions (MI) in enterocytes. MVID is caused by mutations in the MYO5B gene, coding for the Myosin Vb (M5B) motor protein involved in intracellular transport and maintenance of epithelial cell polarity. We examined the effects of loss of M5B in enterocytes of MVID patient and in a CaCo-2 cell model. Method: The expression and localization of cell membrane transporters (CD36, Na/K ATPase) and various cell organelle markers : endosomal Rab GTPases [Rab5, 7, 8, 9, 11] ; early (EEA1) and late endosome (LAMP2), and Golgi (Giantin)) were analyzed using multilabel immunoflourescence and confocal microscopy of a duodenal biopsy from MVID patient and in CaCo-2 cells following M5B si RNA knock-down (Hum Mut 31:1-8,2010). Results: Depletion of M5B in both enterocytes and CaCo-2 cells resulted in disruption of epithelial cell polarity with loss of apical microvilli, formation of MI; mislocalization of transporters as well as aggregation of epitopes for different Rab GTP ases, early and late endosomes. Conclusion: M5B plays a critical role in polarized organization of enterocytes in MVID -a disorder characterized by defective intracellular trafficking and altered endosomal sorting. CaCo-2 cells provide an excellent model to study the pathogenesis of MVID.