Objective: In order to investigate the relationship between the expression of ras gene and the development of hepatocellular carcinoma (HCC). Materials and Methods: The experimental tree shrews were divided into four groups: group A, infected human hepatitis B virus (HBV) and exposed to aflatoxin B1 (AFB1); group B, infected human HBV alone; group C, only exposed to AFB1; group D, use as controls. The serial bioptic liver tissues were detected for ras p21 protein using immunohistochemical method. Results: The total p21protein positive rates in group A, B, C and D were 35.3%, 5.3%, 13.3%, 0, respectively, thus the significant difference were showed between group A and group B (P＜0.05); The HCC incidences in group A, B, C and D were 47.1%, 0, 13.3%, 0, respectively, and there was a significant difference between group A and C (P＜0.05).The incidences of HCC in the animals with and without p21 protein positive in group A were 100% and 18.2%,respectively, and there was a significant difference among them (P＜0.01). Conclusion: HBV and AFB1 play a remarkable synergistic role in the development of HCC; they can enhance the expression of ras gene. The over-expression of ras gene is closely related to pathogenesis of HCC in tree shrews.