Background: Some reports revealed that rapamycin could reactivate HBV infection. However, the mechanism has not been clearly explained. Objectives: In this report, we studied the mechanism by which rapamycin enhances HBV replication and expression by inducing cellular autophagy. Materials and Methods: HepG2.2.15 cells were treated with rapamycin to induce autophagy. Autophagosomes were observed by fluorescence microscopy and transmission electron microscopy. Autophagy marker protein LC3-II/LC3-I was detected by Western blotting. HBV DNA and mRNA were determined by real time PCR and Southern blotting. HBsAg was evaluated by ELISA. Results: In HepG2.2.15 cells, HBV DNA and HBsAg increased when host cells were treated with rapamycin and the effect was reversed by autophagy inhibitor, 3-methyladenine (3-MA). Conclusions: These results indicated a potential explanation for reactivation of HBV infection when patients with hepatitis receive rapamycin.