N-acetylglucosaminyltransferase III (GnT-III), encoded by the MGAT3 glycogene, is thought to be a tumor metastatic suppressor. Our previous studies found that the mRNA expression level of GnT-III decreased in a hepatocyte growth factor (HGF)-induced hepatocellular carcinoma (HCC) epithelial-mesenchymal transition (EMT) model. However, the molecular mechanism of GnT-III expression is unclear. In the present study, we established an HCC EMT model using the classic inducer transforming growth factor-β1 (TGF-β1) and focused on the Smad3 and Erk signaling pathways. Results showed that GnT-III gene expression and its catalytic product bisecting GlcNAc structure decreased and phosphorylation of both Smad3 and Erk1/2 was up-regulated in TGF-β1-treated MHCC97-L cells, while GnT-III mRNA expression and bisecting GlcNAc structure increased after blocking the activities of Smad3 and Erk signaling pathways. These findings suggested that GnT-III gene expression is modulated by the growth factor signaling pathways in the EMT model and that GnT-III may be a potential drug target for HCC treatment.