Alpha-fetoprotein (AFP) is produced principally in fetal liver, gastrointestinal tract and the yolk sac which is temporarily present during embryonic development. AFP is overexpressed in the majority of hepatocellular carcinoma (HCC) and thus offers an attractive target for immunotherapy against this neoplasm. Here, we report that anti-HCC effects were achieved in a therapeutic setting with a DNA vaccine encoding mouse AFP and co-expressing heat shock protein 70 (HSP70) gene. We also demonstrated that this vaccine elicited a marked and highly effective AFP specific CTL response against AFP-positive target cells. This vaccine also induced the prolongation of life span in mice bearing the tumor and the eradication of HCC. It is anticipated that vaccine strategies such as this may contribute to the effective future treatment of hepatocellular carcinoma.