TGF-beta signaling induces epithelial to mesenchymal transition (EMT) and plays an important role in hepatocellular carcinoma (HCC) development. Clinical observations indicate that hepatitis C virus (HCV) chronic infection, which is a major cause of HCC, induces TGF-beta signaling perturbations. Here, we investigate the mechanisms by which HCV nonstructural proteins interfere with TGF-beta signaling, in human hepatoma cell lines expressing HCV subgenomic replicon. A transcriptomic study showed that TGF-beta stimulation of these cells resulted in a protumoral gene expression profile and in up-regulation of EMT-related genes compared to control interferon-treated cells not expressing HCV proteins. We found that the viral protease NS3-4A interacted with SMURF2, a negative regulator of TGF-beta signaling. In cells expressing HCV subgenomic replicon or NS3-4A, TGF-beta stimulation induced an increased expression of SMAD-dependent genes compared to control cells. This enhanced signaling was suppressed by SMURF2 overexpression and mimicked by SMURF2 silencing. In addition, NS3-4A expression resulted in an increased and prolonged TGF-beta-induced phosphorylation of SMAD2/3 that was abrogated by SMURF2 overexpression. Neither NS3-4A protease activity nor SMURF2 ubiquitin-ligase activity was required to affect TGF-beta signaling. Therefore, by targeting SMURF2, NS3-4A appears to block the negative regulation of TGF-beta signaling, increasing the responsiveness of cells to TGF-beta.