Unresectable hepatocellular carcinoma (HCC) lacks effective therapy and entails very poor prognosis. In this study, we have explored a gene-therapeutic approach to stimulate antitumor immunity by adenoviral-mediated transfer of CD40 ligand to treat HCC in rats. In vitro infection of a rat HCC cell line (McA-RH7777) with adenoviral vector expressing CD40 ligand (AdCMVmCD40L) induced CD40L expression in a dose-dependent manner. Expression of CD40L in McA-RH7777 cells did not alter their growth rate in vitro, but it abrogated their tumorigenicity when CD40L-expressing cells were implanted into the liver of syngenic Buffalo rats. In vivo gene therapy of established orthotopic HCC nodules (6.5 mm in diameter) in Buffalo rats by intratumor injection of AdCMVmCD40L vector led to complete tumor eradication and long-term survival in 69.5% of treated animals. Therapy with AdCMVmCD40L induced strong lymphocytic infiltration of the tumoral tissue and increased apoptosis of malignant cells. The observed antitumoral effect was mediated by CD8+ T cells and was associated with increased interleukin (IL)-12 serum levels and enhanced natural killer (NK) activity. Animals that eliminated the tumor after in vivo gene therapy developed protective antitumor immunity being resistant to rechallenge with neoplastic cells. Toxicity of the therapy with AdCMVmCD40L was slight, with only a transient increase in the level of serum transaminases and minor lymphocyte infiltration of normal liver tissue. These data demonstrate that intratumoral administration of AdCMVmCD40L may provide an efficient and safe treatment for HCC.