Chronic infection of hepatitis virus B (HBV) has been proven to be one of the most important risk factors of hepatocellular carcinoma (HCC). HBx has been shown to function in the viral life cycle and the development of HCC. Recently, we have reported that HBx transgenic mice (p21-HBx), generated by gene knockin, develop HCC at the age of 18 months. To further study the function of HBx during the development of HCC in vivo, we performed proteomic analysis of the transgenic and wild-type control mice. The combination of 2-DE and MALDI-TOF MS revealed that proteasome subunits (PSMA6, PSMB4, PSMC2 and PSMD12) were up-regulated in tumor tissues of the p21-HBx transgenic mice. Cathepsin B, ubiquinol-cytochrome C reductase core protein 1 and an ATP-dependent caseinolytic protease, which were involved in the cellular proteolytic process, were also found increased in tumors. The results were confirmed in tumors of transgenic mice and HCCs of human using RT-PCR. All these results suggested that the strengthened ubiquitin-proteasome and lysosomal pathway might contribute to the development of HBx-related HCC.