Autosomal recessive microvillus inclusion disease (MVID) is characterized by an intractable diarrhea starting within the first few weeks of life. The hallmarks of MVID are a lack of microvilli on the surface of villous enterocytes, occurrence of intracellular vacuoles lined by microvilli (microvillus inclusions), and the cytoplasmic accumulation of periodic acid‐Schiff (PAS)‐positive vesicles in enterocytes. Recently, we identified mutations in MYO5B, encoding the unconventional type Vb myosin motor protein, in a first cohort of nine MVID patients. In this study, we identified 15 novel nonsense and missense mutations in MYO5B in 11 unrelated MVID patients. Fluorescence microscopy, Western blotting, and electron microscopy were applied to analyze the effects of MYO5B siRNA knock‐down in polarized, brush border possessing CaCo‐2 cells. Loss of surface microvilli, increased formation of microvillus inclusions, and subapical enrichment of PAS‐positive endomembrane compartments were induced in polarized, filter‐grown CaCo‐2 cells, following MYO5B knock‐down. Our data indicate that MYO5B mutations are a major cause of microvillus inclusion disease and that MYO5B knock‐down recapitulates most of the cellular phenotype in vitro, thus independently showing loss of MYO5B function as the cause of microvillus inclusion disease. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.