Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected, worldwide. Despite the prevalence of infection, gaining a complete understanding of the molecular mechanisms of HBV infection has been difficult since HBV cannot infect common immortalized cell lines. HepG2.2.15, however, is a well-established version of the HepG2 cell line which constitutively expresses HBV. Therefore, comparative proteomics analysis of HepG2.2.15 and HepG2 may provide valuable clues for understanding HBV virus life cycle. In this study, two-dimensional Blue Native/SDS Gel Electrophoresis (2D BN/SDS-PAGE) was utilized to characterize different multi-protein complexes from whole cell lysates between HepG2.2.15 and HepG2. These results demonstrate that two unique protein complexes existed in HepG2.2.15 cells. When these complexes were excised from the gel, subjected to the second-dimension separation, and the proteins sequenced by mass spectrometry, 20 non-redundant proteins were identified. Of these proteins, almost 20% corresponded to heat shock proteins, including HSP60, HSP70 and HSP90. Antibody-based supershift assays were used to verify the validity of the distinct protein complexes. Co-immunoprecipitation assays confirmed that HSP60, HSP70 and HSP90 proteins physically interacted in HepG2 2.15 but not HepG2 cells. We further demonstrated that down regulation of HSP70 or HSP90 by siRNA significantly inhibited HBV viral production but did not influence cellular proliferation or apoptosis. Consistent with these results, a significant reduction in HepG2 2.15 HBV secretion was observed when the HSP90 inhibitor 17-AAG was used to treat HepG2 2.15 cells. Collectively these results suggest that the interaction of HSP90 with HSP70/HSP60 contributes to the HBV life cycle by forming a multi-chaperone machine which may constitute therapeutic targets for HBV-associated diseases.