Transforming growth factor β (TGF-β) exerts an important role in the late steps of carcinogenesis by cooperating with Ras to induce cell motility and tumor invasion. The transcription complex AP-1 has been implicated in the regulation of genes involved in motility and invasion, by mechanisms not yet delineated. We utilized a model of immortalized human hepatocytes (IHH) overexpressing c-Fos (IHH-Fos) or not (IHH-C) to investigate the role of c-Fos on cell motility in response to a prolonged treatment with TGF-β, EGF or a combination of both. Cotreatment with EGF and TGF-β, but neither cytokine alone, induced the conversion of hepatocytes to a fibroblastoid phenotype and increased their motility in Boyden chambers. EGF/TGF-β cotreatment induced a higher effect on ERK phosphorylation compared to TGF-β treatment alone. It also induced an increase in total and phosphorylated Ser178 paxillin, a protein previously implicated in cell motility. This response was inhibited by two specific MEK inhibitors, indicating the involvement of the ERK pathway in paxillin activation. Overexpression of c-Fos correlated with increased cell scattering and motility, higher levels of ERK activation and phospho Ser178 paxillin, increased levels of EGF receptor (EGF-R) mRNA and higher EGF-R phosphorylation levels following EGF/TGF-β cotreatment. Conversely, siRNA-mediated invalidation of c-Fos delayed the appearance of fibroblastoid cells, decreased EGF-R mRNA and downregulated ERK and Ser178 paxillin phosphorylations, indicating that c-Fos activates hepatocyte motility through an EGF-R/ERK/paxillin pathway. Since c-Fos is frequently overexpressed in hepatocarcinomas, this newly identified mechanism might be involved in the progression of hepatic tumors in vivo.