Spinal cord injury (SCI) is a devastating event that often leads to severe disability, and effective treatments for SCI are currently limited. The present study investigated the potential effects and specific mechanisms of melatonin treatment in SCI. Mice were divided into Sham (Sham), Vehicle (Veh), Melatonin (Mel), and Melatonin + 4-phenyl-2-propionamidotetralin (4P-PDOT) (Mel + 4PP) groups based on randomized allocation. The expression of MT2 and the nuclear factor-erythroid 2-related factor 2 (Nrf2)/Keap1 signaling pathways were examined, along with oxidative stress indicators, inflammatory factors and GFAP-positive cells near the injury site. The polarization of microglial cells in different inflammatory microenvironments was also observed. Cell survival, motor function recovery and spinal cord tissue morphology were assessed using staining and Basso Mouse Scale scores. On day 7 after SCI, the results revealed that melatonin treatment increased MT2 protein expression and activated the Nrf2/Keap1 signaling pathway. It also reduced GFAP-positive cells, mitigated oxidative stress, and suppressed inflammatory responses around the injury site. Furthermore, melatonin treatment promoted the polarization of microglia toward the M2 type, increased the number of neutrophil-positive cells, and modulated the transcription of Bax and Bcl2 in the injured spinal cord. Melatonin treatment alleviated the severity of spinal injuries and facilitated functional recovery in mice with SCI. Notably, blocking MT2 with 4P-PDOT partially reversed the neuroprotective effects of melatonin in SCI, indicating that the activation of the MT2/Nrf2/Keap1 signaling pathway contributes to the neuroprotective properties of melatonin in SCI. The therapeutic and translational potentials of melatonin in SCI warrant further investigation.