Objective To develop an efficient, fast and stable establishment method of mouse model of hepatocellular carcinoma. Methods 128 healthy Kunming male mice were equally divided into eight groups: normal group supplied with routine drinking water and food; diethylnitrosamine (DEN)-water group received 16.5 mg/kg DEN in drinking water every day; high-dose DEN-oil groups received orally 16.5 mg/kg of DEN oil solution once, 3 times and 7 times per week, respectively; low-dose DEN-oil group and nano-DEN group received orally 8.25 mg/kg DEN in oil or in nano-vehicle every week, while nano-vehicle group received only an equal amount of nano-vehicle. After twenty weeks, all mice were sacrificed. The paraffin-embedded mouse liver specimens were examined by pathology using HE staining. Results (1) The mice treated with DEN solution showed dry and dull hair, slow movement and slow weight-gain to a varying degree. (2) At the twentieth week, both the DEN-water group and high-dose DEN-oil group showed tumors, and the mortality of high-dose DEN-oil group (once per week) was low. (3) There was no death of mice in the nano-DEN group, and the pathological examination revealed more serious liver injury in the nano-DEN group than in the low-dose DEN-oil group (once per week). Conclusions Oral administration of DEN-oil improved the dietary compliance of mice, and the mortality can be reduced significantly by long-term low frequency DEN administration. Microemulsion preparation of DEN dissolved in oil may enhance the DEN-induced liver damage.