The effect of melatonin (MT) on spinal cord injury (SCI) has attracted increasing research attention. However, the specific role and molecular mechanism of MT on SCI have not been elucidated. An experiment was performed to investigate the effect and molecular mechanism of MT on the neuronal autophagy after SCI and its effect on the recovery of nerve function. The rats were randomly divided into four treatment groups: the SCI+MT+EX527 (SIRT1 inhibitor), SCI+MT, SCI, and sham operation groups. On the 14th day after SCI, MT significantly promoted the recovery of motor function in the hind limbs according to the results of Basso, Beattie, and Bresnahan scores. At the same time, MT treatment resulted in reduced activation of cleaved-caspase-3, cleaved-caspase-9, and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive neurons and increased the survival of motoneurons in the anterior horn of the spinal cord on the 14th day after SCI, which exerted its neuroprotection. Furthermore, western blot and immunofluorescence double staining were performed to verify the molecular mechanism of effect of MT on SCI, and results showed the significantly upregulated expressions of Beclin-1, light chain-3B, SIRT1, p-AMPK proteins in the spinal cord tissue of MT-treated rats on the 14th day after SCI, however, the effect of MT on autophagy was reversed by EX527 (SIRT1 inhibitor), which implied that MT activated autophagy via SIRT1/AMPK signaling pathway after SCI. Similarly, the neuroprotective effects of MT on SCI were also inhibited after the SIRT1/AMPK signaling pathway was suppressed by EX527. Taken together, these results suggest that MT inhibits the apoptosis and activates autophagy of nerve cells after SCI and ultimately exerts the neuroprotective effect by SIRT1/AMPK signaling pathway.