We studied the changes in expression of microRNAs (miRNAs or miRs) and mRNA in normal human bronchial epithelial cells as they differentiate from an undifferentiated monolayer to a differentiated pseudostratified epithelium after 28 days of air-liquid interface (ALI) culture. After 28 days in ALI, the epithelial cells differentially expressed basal, ciliated, and goblet cell markers. Using Affymetrix microarrays, 20 human miRNAs were found to be up-regulated, whereas 35 miRNAs were found to be down-regulated in differentiated cells compared with undifferentiated cells. An analysis of changes in global mRNA expression revealed that 1,201 probe sets demonstrated an 8-fold change (FC) or greater at Day 28 of ALI culture. Of these, 816 were up-regulated and 385 were down-regulated. With differentiation, miR-449a increased (FC, 38.15), and was related to changes in mRNA for cell division cycle 25 homolog A (FC, 0.11). MiR-455 decreased (FC, 0.12) and was related to changes in mRNA for the epithelial cell marker, mucin 1 (FC, 136). Transfection with anti-miR-449 or miR-455-3p resulted in changes in target protein expression (cell division cycle 25 homolog A and mucin 1, respectively), whereas transfection with reporter genes with 3'-untranslated regions of these targets confirmed control of expression through that structure. Therefore, changes in specific miRNAs during human airway epithelial cell differentiation control gene and protein expression important for differentiation.