Abstract: Objective: To explore whether melatonin (MT) could attenuate bupivacaine-induced spinal cord neurotoxicity by inhibiting ferroptosis.Methods: 36 adult male SPF SD rats were randomly divided into three groups:normal saline group (N group), bupivacaine group (B group), and melatonin and bupivacaine co-treated group (BM group).The maximum percentage of anti-injury effect (%MPE) and BBB score were determined on day 0, day 1, day 2 and day 3 of each group for behavioral evaluation and the samples were collected 3 days after administration.The spinal cord tissues at lumbar enlargements were taken for HE and Nissl staining to observe the pathological injury of neurons and the survival number of neurons in the spinal cord tissues of each group, and the mitochondrial ultrastructure change was observed by transmission electron microscopy.The expression levels of reactive oxygen species (ROS) were detected by multiple immunofluorescence, the levels of glutathione (GSH), malondialdehyde (MDA) and iron were detected by ELISA, and the protein expression levels of GPX4 and 4HNE in spinal cord were detected by western blotting.Results: Compared with the N group, MPE% increased, BBB score decreased, spinal cord tissue vacuoles increased, surviving neurons decreased, spinal cord tissue damage was more severe, mitochondrial shrinkage occurred, double membrane density increased, mitochondrial ridge disappeared or was broken, ROS, MDA, and iron levels increased, GSH decreased, and the protein expression of 4HNE increased while GPX4 protein expression decreased (all P< 0.05) in the B group.In comparison with the B group,%MPE decreased, BBB score increased, spinal cord tissue damage was alleviated, spinal cord tissue vacuoles were reduced, surviving neurons increased, mitochondrial damage was improved, ROS, MDA, and iron levels decreased, GSH increased, and the protein expression of 4HNE decreased while the protein expression of GPX4 increased (all P< 0.05) in the BM group.Conclusion: MT can inhibit ferroptosis, thus attenuating the bupivacaine-induced spinal cord neurotoxicity.