Human hepatocellular carcinoma (HCC) is one of the most malignant tumors, being particularly induced by unregulated growth and metastasis, and is a leading cause of death and major health problems in many countries. We report here the identification of 167 differentially expressed proteins between HCC (MHCC97-H) cells and Chang liver cells using enhanced nano-liquid chromatography/mass spectrometry (LC/MS). The most relevant pathways of differentially expressed proteins are involved in cytoskeleton organization, stress defense, and energy homeostasis etc. Moreover, of the identified proteins, there are 59 known or putative membrane-associated proteins with multitransmembrane domains confirmed by bioinformatic analysis. These proteins may be associated with cancer, reflecting tumorigenesis of HCC, and would be useful for the development of diagnostic and subsequently pharmaceutical targets of HCC. In addition, we identify a total of 41 proteins that are found to be up- or down-regulated following tanshinone IIA treatment for MHCC97H cells in a time-depended manner. Also, several proteins that are involved in actin cytoskeleton and stress resistance are mainly down-regulated, whereas proteins associated with cell redox homeostasis, mitochondrial, and microtubule-based movement are identified as mostly up-regulated after the treatment. Determination of functional roles of those differentially expressed proteins will enable further understanding of the mechanism of HCC tumorigenesis and exploration of new drugs for therapeutic intervention.