Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide. The initial hepatocellular alterations that precede the appearence of HCC include chronic viral hepatitis/cirrhosis, foci of phenotypically altered hepatocytes and, subsequently, dysplastic hepatocytes that form foci and nodules. These changes cause a discrepancy in the microenvironment of liver cells, which may result in changes in the protein expression profile of the cells. The aim of the present study was to investigate differences between the protein expression profiles at various stages of liver disease in order to better understand the mechanisms of HCC and to identify potential biomarkers for its early diagnosis. The proteins of specific cells were obtained from HCC tissue sections and pre-cancerous lesions using a manual microdissection technique, and were investigated by a two dimensional gel electrophoresis (2-DE) MALDI-TOF MS proteomics approach. Select identified proteins were reconfirmed by immunohistochemistry. A total of 95 differentially expressed proteins, with an over 2-fold disparity in expression levels between cells of varying morphology during the stages of hepatocarcinogenesis, were detected by 2-DE. Among these 95 proteins, 80 were determined to be involved in numerous cell functions, including cell growth and proliferation, protein synthesis and metabolism, apoptosis and signal transduction. These identified proteins, which include stratifin (14-3-3), transgelin 2, heat-shock protein (HSP)70, HSP27, manganese superoxide dismutase, prohibitin, DJ1, α-enolase, peroxiredoxin 6, aldo-keto reductase family member B10, phosphoglycerate kinase 1, α-1-antitrypsin and nm23-H1, may play a role in the development of HCC. Protein expression profiles differed markedly between the HCC tissue samples and pre-cancerous lesions, suggesting that alterations in protein expression occurred frequently during the process of hepatocarcinogenesis. Analysis of the differential expression of proteins related to the development of HCC may help elucidate the molecular mechanisms of the disease. These proteins may also serve as candidate biomarkers for early HCC diagnosis.