Chronic hepatitis B virus (HBV) infection is a major cause of liver fibrosis, eventually leading to cirrhosis and hepatocellular carcinoma. Although the involvement of the X protein of HBV (HBx) in viral replication and tumor development has been extensively studied, little is known about its possible role in the development of fibrosis. In this work we show that expression of HBx in hepatocytes results in paracrine activation and proliferation of hepatic stellate cells (HSCs), the main producers of extracellular matrix proteins in the fibrotic liver. Both human primary HSCs and rat HSCs exposed to conditioned medium from HBx-expressing hepatocytes showed increased expression of collagen I, connective tissue growth factor, α smooth muscle actin, matrix metalloproteinase-2, and transforming growth factor-β (TGF-β), together with an enhanced proliferation rate. We found that HBx induced TGF-β secretion in hepatocytes and that the activation of HSCs by conditioned medium from HBx-expressing hepatocytes was prevented by a neutralizing anti-TGF-β antibody, indicating the involvement of this profibrotic factor in the process.Conclusion:Our results propose a direct role for HBx in the development of liver fibrosis by the paracrine activation of stellate cells and reinforce the indication of antiviral treatment in patients with advanced HBV-related chronic liver disease and persistent liver replication.