Objective: To investigate the inhibitory effect and mechanism of recombinant analgesic-antitumor peptide （rAGAP） enhanced 5-fluorouracil（5-FU） on H22 hepatoma. Methods: The models of H22 hepatoma in mice were established through ascites inoculating H22 cells suspension into mice in right armpits， and randomly divided into 4 groups. rAGAP group was given 0.03 mg/kg IP rAGAP， 5-FU group 10 mg/kg IP 5-FU， United group 0.03 mg/kg 5-FU and 10 mg/kg IP rAGAP， Model group used equal volume of normal saline intraperitoneal injection for 3 weeks. After that， the tumor tissue were sampled， the tumor weight and tumor inhibition rate were detected. The expressions of PI3K， AKT， and PTEN were detected by Western blot method. Results: Compared with Model group， mice tumor weight of other groups was significantly lower （P<0.05）; tumor suppressor rate of United Group was obviously higher than that of rAGAP group and 5-FU group （P<0.05）. Compared with Model group， PI3K and p-Akt expression of other groups were significantly decreased， but PTEN expression was significantly increased （P<0.05）; compared with 5-FU group， rAGAP group， PI3K and p-Akt expression of United group were significantly reduced， but PTEN expression was significantly increased （P<0.05）. Conclusion: rAGAP can enhance the inhibitory effect of 5-FU on H22 hepatoma， its mechanism may be related to the activity of PI3K/AKT/PTEN signaling pathway， and then inhibition the proliferation of H22 hepatoma.