Objective To explore the effect and mechanism of midazolam inducing ferroptosis in cervical cancer cells. Methods Human cervical cancer HeLa cells were cultured in vitro and divided into 5 groups. Low/ medium/high dose midazolam groups were injected with 5 doses，respectively 5，10，20 μmol/L midazolam，high⁃ dose midazolam+nuclear factor E2 related factor 2 activator group injected with 20 μmol/L midazolam and 80 nmol/L Bardoxolone，as well as normal cultured HeLa cells，were used as control groups for further cultivation for 24 hours. CCK⁃8 and clone formation assay were used to detect cell proliferation；PI staining was used to detect cell death； transmission electron microscopy was used to observe mitochondrial morphological changes ；kits were used to detect the levels of iron，reactive oxygen species（ROS），glutathione（GSH），malondialdehyde（MDA）；Western Blot was used to detect the protein expressions of Nrf2/heme oxygenase⁃1（HO⁃1）signaling pathway⁃related proteins and GSH peroxidase 4（GPX4）. Results Compared with the Control group，the L⁃midazolam group，M⁃midazolam group，and H⁃midazolam group showed a decrease in HeLa cell viability，a decrease in clone count，an increase in cell mortality，and a significant ferroptosis feature in mitochondria. The levels of iron，ROS，and MDA in cells increased，while GSH levels and GPX4，nuclear Nrf2，and HO⁃1 protein expression decreased. The H⁃midazolam group showed more significant changes（P < 0.05）. Compared with the H ⁃midazolam group，the H ⁃midazolam+ Nrf2 activator group showed an increase in HeLa cell viability，increased clone count，decreased cell mortality， reduced mitochondrial iron death damage，decreased levels of iron，ROS，and MDA in cells，and increased levels of GSH and GPX4，Nrf2，and HO⁃1 protein expression（P < 0.05）. Conclusion Midazolam can inhibit the prolif⁃ eration of HeLa cells and induce ferroptosis in a dose dependent manner，which may be achieved by inhibiting the Nrf2/HO⁃1 signaling pathway.