Abstract: Objective: To observe whether insulin can enhance the chemotherapeutic response of 5-fluorouracil (5-FU) in transplanted H22 liver cancer in nude mice and to investigate the underlying mechanism. Methods: A total of 60nude mice with transplanted H22 liver cancer were randomly divided into 6 groups with 10 in each group as follows: thecontrol group, the group that received insulin (0.09U/20g) alone, the group that received 5-FU alone, the group that re-ceived high-dose insulin(0.09U/20g) plus 5-FU, the group that received medium-dose insulin(0.06U/20g) plus 5-FU, andthe group that received low-dose insulin (0.03U/20g) plus 5-FU. The expression of cyclin D was detected in tumor tissuesby immunohistochemistry, and the percentage of cells in each phase of the cell cycle was observed by flow cytometry. Results: Compared with the 5-FU group, the insulin plus 5-FU groups had greater reductions in tumor weight. In thehigh-dose insulin(0.09U/20g) plus 5-FU group, the tumor weight was significantly reduced and the rate of inhibition of tu-mor growth was 51.35%, higher than that in the group that received 5-FU alone (27.93%, P<0.05). The expression rate ofcyclin D was 62% in the group that received insulin (0.09U/20g) alone. This rate was higher than that seen in the controlgroup(30%, P<0.05). The percentage of cells in G 1 phase and S phase in the insulin-treated(0.09U/20g) group was 53.87%and 6.13%, respectively. Compared with the control group, the insulin-treated(0.09U/20g) group showed a decrease in thepercentage of cells in G 1 phase and an increase in the percentage of cells in S phase(P<0.05). Conclusion: Insulin can en-hance the chemotherapeutic response of 5-FU on transplanted H22 liver cancer. Insulin can induce S phase arrest in can-cer cells by increasing the expression of Cyclin D, sensitizing tumor cells to the S phase-specific anti-tumor drug 5-FU.