Loss of function mutations in the actin motor Myosin Vb (Myo5b) lead to Microvillus Inclusion Disease (MVID) and death in newborns and children. MVID results in secretory diarrhea (SD), brush border (BB) defects, villus atrophy and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl^-) and sodium (Na⁺) is unknown. The current study used Myo5b loss of function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal gSTED imaging, immunohistochemical staining, TEM, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression and function of the major BB ion transporters NHE3 (Na⁺), CFTR (Cl^-) and SLC26A3 (DRA) (Cl^- /HCO3^-) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory crypt like enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BBM of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation and a crypt-like phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3 and DRA contribute to Cl^- and Na+ stool loss in MVID diarrhea.