Hepatocellular carcinoma (HCC) is the fifth most frequent cause of cancer deaths in males and was the third most frequent cause of cancer deaths in 2007 throughout the world. The incidence rate is 2-3 times higher in developing countries than in developed countries. Animal models have enabled study of the mechanism of HCC and the development of possible strategies for treatment. Diethylnitrosamine (DEN) is a representative chemical carcinogen with the potential to cause tumors in various organs, including the liver, skin, gastrointestinal tract, and respiratory system. Specifically in HCC, DEN is a complete carcinogen. Many lines of evidence have demonstrated a relationship between carcinogenesis and cell cycle regulation. In this study we found that cell cycle regulatory proteins were critically involved in cancer initiation and promotion by DEN. Cyclin D1, cyclin E, cdk4, and p21(CIP1/WAF1) are factors whose expression levels may be useful as criteria for the classification of hepatic disease. In particular, cdk4 had a pivotal role in the transition to the neoplastic stage. In conclusion, we suggest that changes in the level of cdk4 may be useful as a biomarker for detection of HCC.