The ability of non-small cell lung cancer (NSCLC) cells to invade and metastasize is associated with epithelial-to-mesenchymal transition (EMT). The process of EMT is, at least in part, regulated by microRNAs. However, it is unknown whether microRNAs regulate EMT in cancer stem-like cells (CSLCs), or which microRNAs are involved. In the present study, we compared microRNA expression in A549 cells, TGF‑β1-treated A549 cells, CSLCs characterized by the CD133+/CD326+ phenotype, and TGF‑β1-treated CSLCs. We found that miR‑181b‑5p expression was upregulated by TGF‑β1. Moreover, the overexpression of the miR‑181b‑5p in A549 cells and CD133+/CD326+ cells resulted in the downregulation of the E-cadherin and increased invasion and metastasis in vitro and in vivo. Accordingly, the knockdown of miR‑181b‑5p partially restored E-cadherin expression. These results suggest that miR‑181b‑5p regulates TGF‑β1-induced EMT by targeting E-cadherin not only in normal A549 cells but also in CD133+/CD326+ cells which have characteristics of CSLCs. Thus, miR‑181b‑5p represents a new therapeutic target in NSCLC.