Epithelial-mesenchymal transition (EMT), the epithelial cells transdifferentiation into the mesenchymal cells, has been involved in cancer metastasis. Nannocystin ax (NAN) is a cyclodepsipeptide initially isolated from Myxobacterial genus, Nannocystis sp. with anticancer activities. This study was designed to explore the effect of NAN on TGF-β1-induced EMT in lung cancer cells. The morphological alteration was observed with a microscope. Western blotting and immunofluorescence assays were used to detect the protein expression and the localization. The adhesion and migration were evaluated by adhesion assay and wound healing assay. The mRNA expression of TGF-β receptor type I (TβRI) was determined by real-time PCR. NAN significantly restrained TGF-β1-induced EMT morphological changes, the protein expression of E-cadherin, N-cadherin, and Vimentin, etc. TGF-β1 activated phosphorylation and nuclear translocation of Smad2/3 were inhibited by NAN. Furthermore, NAN suppressed adhesion and migration triggered by TGF-β1. In addition, NAN significantly down-regulated TβRI on the transcriptional level directly. In summary, these results showed that NAN restrained TGF-β1-induced epithelial-mesenchymal transition, migration, and adhesion in human lung cancer cells. The underlying mechanism involved the inhibition of Smad2/3 and the TβRI signaling pathway. This study reveals the new anticancer effect and mechanism of NAN.