Acute liver failure (ALF) of infectious origin results from massive Fas-mediated hepatocyte apoptosis. To cure Fas-induced ALF in mice, we have designed a noninvasive procedure for intrahepatic transfer of a plasmid that encodes a molecule inhibiting Fas-Fas ligand interaction. For that purpose, naked pDNA encoding green fluorescent protein (GFP) or the Fas-Fc chimeric protein was transferred into mice by the biliary route. Ten percent of hepatocytes expressed GFP. After pFas-Fc transfer, about 40 ng of Fas-Fc protein per milliliter could be detected in sera from day 4 to day 28. Serum recombinant Fas-Fc could neutralize Fas-induced cell death in vitro. Furthermore, pFas-Fc biliary transfer efficiently protected mice against Fas-mediated ALF, because survival rates (p < 0.01), serum transaminase activities (p < 0.05), and histological data (p < 0.02) were improved versus control pTNFR-Fc-transfected mice. In conclusion, naked pDNA encoding Fas-Fc is efficiently expressed by hepatocytes after biliary gene transfer in mice. This method, devoid of virus-related risks, could be considered for the treatment of Fas-mediated ALF in humans.