Objective We investigated the effect of mothers against decapentaplegic homolog 6(Smad6) on the migration and epithelial-mesenchymal transition of human glioma cell lines by targeting microRNA-186 (miR-186). Methods The expression of miR-186 and Smad6 at the mRNA and protein level ( for Smad6) was determined by RT- qPCR and / or Western blot in astrocytes and glioma cells. The cells were divided into four groups: a blank control group (U251), a mimic group (miR-186 mimics), a plasmid group (pc-smad6), and a co-transfection group (miR-186 mimics + pc-smad6). The expression of Smad6, E-cadherin, and Vimentin at the mRNA and protein level was determined by RT- qPCR and Western blot in each group. Migration ability was measured using a wound healing experiment. Results Compared with astrocytes, the expression of miR-186 in human glioma cell line U251 decreased significantly, while the expression of Smad6 mRNA increased significantly (P <0.05). Compared with the control group, the expression level of the Smad6 protein in the miR-186 mimic group decreased significantly, while the level of the Smad6 protein in the pc-smad6 group increased significantly (P <0.05). Compared with the pc-smad6 group, the expression level of the Smad6 protein in the miR-186 mimics + pc-smad6 group significantly decreased (P <0.05). Compared with the control group, the migration ability of the miR-186 mimics group decreased significantly, while the pc-smad6 group’s migration ability was enhanced. Compared with the pc-smad6 group, the migration ability of the miR-186 mimics + pc-Smad6 group decreased significantly (P <0.05). Compared with the control group, E-cadherin expression in the miR-186 mimics group increased significantly, while Vimentin protein expression decreased significantly (P <0.05).The expression of the two molecules in the pc-smad6 group was the opposite (P <0.05). Compared with the pc-smad6 group, vimentin expression in the miR-186 mimics + pc-smad6 group decreased significantly, while E-cadherin expression increased significantly ( P <0.05). Conclusions miR-186 may regulate migration and epithelial-mesenchymal transition of glioma cells by targeting Smad6.