Hepatocellular carcinoma (HCC) in China is mostly Hepatitis B virus infection related. The antitumor efficacy of HBsAg gene-modified dendritic cells (DC) has been widely tested both in vitro and in vivo. In this study, we analyzed whether adenoviral vector mediated HBsAg expression would alter cell surface phenotype or autologous T cell stimulating function of mature DCs. Further, the anti-tumor efficacy of pAd-HBsAg-DC-based vaccine was evaluated in mice bearing HBsAg expressing HCC. We also tested whether β-glucosylceramide (β-GC) would enhance the anti-tumor activity of pAd-HBsAg-DC. Results revealed that pAd-HBsAg-DC expressed and secreted HBsAg, while maintaining phenotypic characteristics of mature DCs. Vaccination with pAd-HBsAg-DC conferred specific therapeutic antitumor immunity to animal model bearing HBsAg expressing HCC. The application of β-GC activated mice hepatic NKT cells and enhanced the antitumor activity of pAd-HBsAg-DC. Most importantly, in vivo results showed that the inhibiting effect of pAd-HBsAg-DC vaccination on tumor growth was more significant when applied before tumor inoculation, suggesting that genetically modified DC based therapeutic cancer vaccine may achieve the most optimized antitumor effect when applied before tumor onset, and β-GC may serve as a potent innate immune enhancer for augmenting the antitumor effect of pAd-HBsAg-DC vaccine.