The subcellular proteome strategy can complement the separation power of two-dimensional electrophoresis, a step for subcellular fractionation is introduced before electrophoresis is conducted, and more proteins will be displayed in two-dimensional gels. A comparative analysis of proteomic profiling of mitochondria and nuclei was conducted between hepatoma cell and hepatocyte, in order to find more information involved in cancer development. The cultured hepatoma cell line QGY-7703 and hepatocyte line LO2 were used as research models in this work. Subcellular fractionation for mitochondrion and nucleus were done by ultracentrifugation, then two-dimensional electrophoresis was applied to the separation of mitochondrial and nuclear proteins, imaging analysis for the selection of differentially-expressed protein spots, and MALDI-TOF-MS for the identification of proteins. 54 spots from electrophoresis gels were selected as differentially-expressed protein for MS analysis which resulted in identification for 22 proteins. Among the 22 differentially-expressed proteins, 17 show a up-regulated expression and 5 show a down-regulated expression. These differentially-expressed proteins found in this work have a wide coverage of functions which are related to energy metabolism, cytosheleton, protein biosynthesis, pre-mRNA splicing and processing, apoptosis regulation. These results imply that cancer cell has experienced a fundamental change in structure and metabolism pattern.