Objective Moxibustion is a classic traditional Chinese medicine treatment that combines physical therapy (hyperthermia) and chemical therapy. It has been shown to have an inhibitory effect on a variety of cancers, but the cellular biological mechanism is still unclear. This study aims to study the effect of hyperthermia on astrocytic cancer cell line U251, and to reveal the effect of moxibustion on cancer cells through hyperthermia.Methods The morphological changes of U251 cells treated with hyperthermia at 42℃ for 108 h were recorded by microphotography. The changes of total cell number, live cell number and live cell proportion were detected by cell counting after 3 d of hyperthermia to evaluate the effects of hyperthermia on cell proliferation and death. The effect of hyperthermia on cell apoptosis was evaluated by detecting Annexin V and Caspase-3.Results Hyperthermia significantly inhibited cell proliferation and increased cell death. Compared with the control group, the U251 cells treated with hyperthermia had obvious nuclear pyknosis, and Annexin V and PI markers were significantly increased, indicating that the cell death caused by hyperthermia was mainly caused by Caspase related apoptosis.Conclusion This study demonstrated that hyperthermia can inhibit cell proliferation and induce Caspase related apoptosis in astrocytic cell line U251.