Rab11a is a key component of the apical recycling endosome that aids in the trafficking of proteins to the luminal surface in polarized epithelial cells. Utilizing conditional intestinal epithelial cell-specific Rab11a knockout mice and human colonic epithelial CaCo2-BBE cells with stable Rab11a knockdown, we examined the molecular and pathological impact of Rab11a deficiency on the establishment of apical cell polarity and microvillus morphogenesis. We demonstrate that loss of Rab11a induced alterations in enterocyte polarity, shortened microvillar length and the formation of microvilli along the lateral membranes. Rab11a deficiency in enterocytes altered the apical localization of Syntaxin 3. These data affirmed the role of Rab11a in apical membrane trafficking and the maintenance of apical microvilli in enterocytes.