Alveolar epithelial type II cells (AEC II) are multifunctional cells in the lung. In addition to the secretory and synthetic functions, they have progenitor capacities. When alveolar epithelial type I cells (AEC I) are damaged under various disease conditions, cuboidal AEC II proliferate and trans‐differentiation into squamous ACE I, which helps to restore the normal air‐blood barrier. However, the molecular mechanisms of this trans‐differentiation process remain largely unknown. MicroRNAs are a recently discovered class of small noncoding RNA, which negatively regulate the expression of target genes at the post‐transcriptional level. Through microRNA microarray and real‐time PCR analyses, we found that microRNA‐375 (miR‐375) was significantly decreased during the trans‐differentiation of AEC II to AEC I. miR‐375 was expressed higher in adult lungs that fetal lungs and appeared to be enriched in AEC II. The Wnt/β‐catenin pathway was activated during alveolar epithelial cell differentiation. Overexpression of miR‐375 prevented the trans‐differentiation of AEC II to AEC I and inhibited the Wnt/β‐catenin signaling. In conclusion, our study shows that miR‐375 regulates the trans‐differentiation of AEC II to AEC I through the Wnt/β‐catenin signaling pathway.