Objective To assess the clinical significance of histone deacetylase 6 (HDAC6) expression in nonsmallcell lung cancer (NSCLC) and determine the relationship between HDAC6 and the epithelial-mesenchymal transition(EMT). Methods Tumor and noncancerous peritumoral lung tissues (controls) were collected from 52 patients withNSCLC and analyzed for HDAC6 and E-cadherin expression using immunohistochemistry. Correlation analyses betweenHDAC6, E-cadherin and clinicopathological parameters were performed using χ2 tests. In vitro studies were conducted intransforming growth factor (TGF)-β1-induced A549 cells to determine the effect of HDAC6 overexpression on molecularmarkers of EMT. Results Immunohistochemical analysis revealed significantly elevated HDAC6 expression in NSCLCtissues, which correlated with the differentiation stage and lymph node metastasis. Moreover, HDAC6 expression wasinversely correlated with that of E-cadherin, an EMT marker. Treatment with an HDAC6 inhibitor and signaling inhibitors ofRho-associated protein kinase ( ROCK) or extracellular signal-regulated kinases ( ERK) significantly reduced theproliferation, migration and invasion of TGF-β1-induced A549 cells. Furthermore, western blot analysis indicated that TGF-β1 induced EMT in A549 cells, which was manifested by upregulated E-cadherin and vimentin and downregulated α-smooth muscle actin (α-SMA). Signaling inhibitors against HDAC6, ROCK and ERK reversed the effects of TGF-β1treatment. Conclusions Our data suggest that HDAC6 plays an important regulatory role in NSCLC tumorigenesis andprogression and is closely related to tumor EMT and regulation of ROCK and ERK signaling. Targeting HDAC6 activity using HDAC6, ERK1/2 or ROCK inhibitors may be a potential therapeutic option for NSCLC.