Objective To study the role of vagusism on diethytlnitrosamine (DEN)-induced hepatocellular carcinoma in rats and to explore its mechanism.Methods The rat vagus nerve was labeled with 1,1'-dioctadecyl-3,3,33 -tetramethylindocarbocyanine perchlorate (DiI) tracer.The rats were divided into three groups:cancer induced rat models (cancer-induced group,n =20),cancer-induced and vagal detachment models (combined group,n =20) and normal rats (negative group,n =20),HE staining was performed on the liver tissue biopsied from the three groups of rats.The pathological grading score was evaluated and statistically analyzed.Immunohistochemistry (IHC) staing was used to determine the expression of vagal neurotransmitters and related receptors in liver tissue.Results The optimal concentration (1 mg/ml) and fixation duration with formalin (4 days) of DiI tracer marker vagal nerve demonstrated superior distribution and density of vagus nerve in the liver tissue.The incidence of hepatocellular carcinoma was significantly higher in the cancer-induced group (75％) than combined group (15％),the difference was statistically significant (P ＜ 0.05).IHC staining results of acetllcholine showed a significant difference between cancerinduced group (9.10) and combined group (2.30) or negative group (2.95),the difference was statistically significant (P ＜ 0.05).The M1 receptor expression was significantly higher in cancer-induced group (6.00) than combined group (2.30) and negative group (2.55),the differences were statistically significant (P ＜ 0.05).There was no significant difference of M2 receptor expression among the cancer-induced group (1.30),combined group (1.25) and negative group (1.35) (P ＞ 0.05).The M3 receptor expression detected with IHC staining shown there were statistical differences among cancer-induced group (8.95),combined group (6.30) and negative group (3.60) (all P ＜ 0.05).Conclusion The vagus nerve may play an important role in the development of DEN-induced hepatocellular carcinoma in rats.