AbstractBackgroundForkhead box L2 (FOXL2) has been recognized as a transcription factor in the progression of many malignancies, but its role in non‐small cell lung cancer (NSCLC) remains unclear. This research clarified on the role of FOXL2 and the specific molecular mechanism in NSCLC.MethodsRNA and protein levels were detected by quantitative real‐time polymerase chain reaction (qRT–PCR) and western blotting assays. Cell proliferation was examined by cell counting kit‐8 (CCK‐8) and clonogenic assays. Transwell and wound healing assays were used to detect cell invasion and migration. Cell cycle alterations were assessed by flow cytometry. The relationship between FOXL2 and miR‐133b was verified by dual‐luciferase reporter assays. In vivo metastasis was monitored in the tail vein‐injected mice.ResultsFOXL2 was upregulated in NSCLC cells and tissues. Downregulation of FOXL2 restrained cell proliferation, migration, and invasion and arrested the cell cycle of NSCLC cells. Moreover, FOXL2 promoted the epithelial–mesenchymal transition (EMT) process of NSCLC cells by inducing the transforming growth factor‐β (TGF‐β)/Smad signaling pathway. miR‐133b directly targeted the 3′‐UTR of FOXL2 and negatively regulated FOXL2 expression. Knockdown of FOXL2 blocked metastasis in vivo.ConclusionsmiR‐133b downregulates FOXL2 by targeting the 3′‐UTR of FOXL2, thereby inhibiting cell proliferation, EMT and metastasis induced by the TGF‐β/Smad signaling pathway in NSCLC. FOXL2 may be a potential molecular target for treating NSCLC.