Objective To explored the effects of fentanyl on cell proliferation of H1299 cells, Methods After treating H1299 cells with different concentrations of fentanyl (0.001, 0.010, 0.100, 1.000 μM) for 12, 24, 48, 72 h, cell viability was detected by CCK-8 assay; the rate of cell apoptosis was determined by DAPI staining; the expression levels of Bax, Bcl-2, p-AKT and AKT protein were measured by Western blotting; Caspase-3 activity was determined by Caspase-3 activity assay kit. Results Compared with the control group, fentanyl obviously inhibited the viability of H1299 cells in a dose and time dependent way. Moreover, treatment with different concentrations of fentanyl(0.001, 0.010, 0.100, 1.000 μM) for 12, 24, 48, 72 h, the apoptosis rate of H1299 cells were significantly increased, The level of Bcl-2 protein reduced the level of Bax protein, and the activity of Caspase-3 in H1299 cells were increased after treatment with fentanyl (0.010, 0.100, 1.000 μM) for 48 h, Furthermore, fentanyl markedly inhibited p-AKT/AKT activity of H1299 cells. Conclusions Fentanyl can inhibit cell proliferation and promote cell apoptosis of human lung cancer, and its mechanism may be related to inhibition of AKT activation ,