Airway epithelial cells cultured at an air–liquid interface bear many hallmarks of in vivo cells and are used extensively to study the biology of the lung epithelium. Because miRNAs regulate many cellular functions, we postulated that miRNA profiling would provide an unbiased assessment of the effects of in vitro culturing. RNA was extracted from primary airway epithelial cells either immediately after cell procurement (in vivo condition) or after air–liquid interface culture was established (in vitro condition). We assessed 742 miRNAs and determined differential expression between in vivo and in vitro conditions. Air–liquid interface culturing of airway epithelial cells caused widespread changes in miRNA expression. A similarly extensive alteration in gene expression was observed in an independent set of publicly available microarray data. We integrated miRNA and gene expression results to identify culture-induced differences in transcriptional programs (including several involved in epithelial injury and repair). Air–liquid interface cultures are useful models for studying airway biology, but the present findings indicate that, despite phenotypic similarities with primary cells, these culture systems profoundly perturb miRNA and gene expression. Studies of lung epithelium based on in vitro culture should therefore be designed and interpreted with an appreciation of the limitations of air–liquid interface culture systems.