Cytotoxic T lymphocytes and their granule components, such as perforin and granzyme, play an important role in the defense of hepatic infections caused by different pathogens. Moreover, it has been shown in vitro that hepatocytes can initiate cell death via a perforin-dependent mechanism. Although it is well known that hepatocellular apoptosis in D-galactosamine/lipopolysaccharide (D-Gal/LPS)-associated liver failure is mediated by TNF-dependent Fas/FasL cytotoxicity, there is no information on the role of perforin-mediated mechanisms in vivo. Therefore, we studied whether the cytolytic perforin/granzyme pathway contributes to the D-Gal/LPS-associated hepatotoxicity. Perforin knock out (Pko) mice showed significantly higher hepatic TNF and IL-6 mRNA expression as well as plasma TNF and IL-6 concentrations within the first hour upon D-Gal/LPS challenge when compared to perforin wild type (Pwt) mice. At 6 hours upon D-Gal/LPS challenge Pko mice further presented with higher transaminase release and onconecrotic tissue damage, while hepatocellular apoptosis and caspase-3 cleavage remained unaffected by the perforin deficiency. Pretreatment with a recombinant human TNF-receptor fusion protein attenuated necrotic and apoptotic tissue damage, and reduced plasma transaminase activities as well as cytokine release, thereby preventing acute liver failure in Pko mice as effective as in Pwt mice. These data do not only confirm the significance of TNF as distal mediator of hepatic injury in this model, but simultaneously reveal a contribution of a perforin-dependent immunoregulation, limiting the D-Gal/LPS-induced overwhelming cytokine release and onconecrotic tissue injury.