The majority of persons with chronic hepatitis C virus (HCV) infection develop liver fibrosis. Transforming growth factor (TGF)-β1 plays a pivotal role in the pathogenesis of post-inflammatory liver scarring. To clarify the influence of HCV infection on liver fibrosis, a reporter assay was used to investigate the effect of viral proteins on TGF-β1 expression in human hepatoma cells. Of all HCV proteins investigated (core, E1/E2/p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), only the core protein activated the TGF-β1 promoter and upregulated TGF-β1 expression measured by an RNase protection assay. Bases −376 to −331 bp in the promoter region of TGF-β1 are responsible for upregulation by HCV core protein, and the nuclear protein that binds to this region increased with the stimulation of HCV core protein. Blocking the mitogen-activated protein kinase pathway prevented upregulation of TGF-β1 by HCV core protein. The immunological response is supposed to be a major factor to cause the secretion of TGF-β1 from non-parenchymal cells, but the results suggest that the HCV core protein expression may upregulate directly TGF-β1 transcription in parenchymal cells and suggest a new paradigm for exacerbation of liver fibrosis by HCV infection. J. Med. Virol. 72:52–59, 2004. © 2004 Wiley-Liss, Inc.