Objectives We evaluated the effects of miR-140-3p on EMT, cellular migration, and invasion in TGF-beta 1 treated human OS cells. Human fresh OS tissue and normal bone tissue specimens were gathered from 42 patients (29 male and 13 female, 11 to 24 years of age with a mean age of 17.5 +/- 2.3 years) diagnosed with OS by pathology. By targeting TRAF6, miR-140-3p inhibits TGF-beta 1-induced human osteosarcoma epithelial-to-mesenchymal transition, migration, and invasion. Results In this study, we found microRNA (miR)-140-3p to be down-regulated and tumor necrosis factor receptor-associated factor 6 (TRAF6) to be up-regulated in patient OS samples. Lower levels of miR-140-3p and higher levels of TRAF6 were found in the advanced Enneking stage of OS. Furthermore, both mRNA and protein levels of TRAF6 were negatively associated with miR-140-3p mRNA expression in human OS tissue. TRAF6 was verified as a direct target of miR-140-3p in TGF-beta 1-treated human U2OS cells. Further, a miR-140-3p mimic dramatically inhibited while a miR-140-3p inhibitor enhanced TGF-beta 1-induced epithelial-to-mesenchymal transition, migration, and invasion of U2OS cells. Small interfering RNA was found to silence TRAF6 and to partly reverse the effects of the miR-140-3p inhibitor on TGF-beta 1-treated U2OS cells in vitro. Conclusion These results demonstrate miR-140-3p to function as a tumor inhibitor of human OS cells by decreasing TRAF6 expression. miR-140-3p and TRAF6 may be valuable and novel biomarkers for diagnosis and treatment of OS.