Salinomycin was recently identified as an antitumor drug for the treatment of several types of solid tumors. However, the effects of salinomycin on the migratory and invasive properties of non-small cell lung cancer (NSCLC) are unclear. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of salinomycin on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In A549 and H460 NSCLC, salino-mycin strongly blocked the enhancement of cell migration by TGF-β1 induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1 mediated AMPK/SIRT signaling activity. We demonstrated that salinomycin was effective in preventing TGF-β1-induced EMT, as indicated by upregulation of E-cadherin, and downregulation of mesenchymal markers and transcription factors. Moreover, salinomycin could inhibit TGF-β1-enhanced migration and invasion. MMP-2 and MMP-9 downregulation enhanced the reversal of TGF-β1-induced EMT by salinomycin. In contrast, AMPK/SIRT upregulation promoted TGF-β1-induced EMT. Taken together, these results indicate that salinomycin can inhibit TGF-β1-induced EMT and suppress lung cancer cell migration and invasion via downregulation of MMP-2 and MMP-9. Our findings implicate overexpressed AMPK/SIRT1 as a potential therapeutic target to reverse TGF-β1-induced EMT and to prevent lung cancer cell migration and invasion.