Hepatitis B virus (HBV) infection is a worldwide health problem and may develop to liver fibrosis, cirrhosis, and even hepatocellular carcinoma. To investigate the global proteome responses of liver-derived cells to HBV infection and IFNα treatment, 2-DE and MS-based analysis were performed to compare the proteome changes between HBV stably transfected cell line HepG2.2.15 and its parental cell line HepG2, as well as HepG2.2.15 before and after IFNα treatment (5000 IU/mL for 72 h). Compared to HepG2, 12 of 18 down-regulated and 27 of 32 up-regulated proteins were identified in HepG2.2.15. After IFNα treatment, 6 of 7 down-regulated and 11 of 14 up-regulated proteins were identified. Differentially expressed proteins caused by HBV infection were involved with cytoskeletal matrix, heat shock stress, kinases/signal transduction, protease/proteasome components, etc. Prohibitin showed a dose-dependent up-regulation during IFNα treatment and might play a potent role in anti-HBV activities of IFNα by enhancing the crossbinding p53 expression to achieve the apoptosis of HBV infected liver cells. Down-regulation of interferon-stimulated gene 15 (ISG15) in HepG2.2.15 and recovery by IFNα suggested its relationship with IFNα's anti-HBV effect.