Spinal cord injury (SCI) disturbs the autonomic nervous system and induces dysfunction in multiple organs/tissues, such as the gastrointestinal (GI) system. The neuroprotective effects of melatonin in SCI models have been reported; however, it is unclear whether the beneficial effects of melatonin are associated with alleviation of gut dysbiosis. In this study, we showed that daily intraperitoneal injection with melatonin following spinal cord contusion at thoracic level 10 in mice improved intestinal barrier integrity and GI motility, reduced expression levels of certain proinflammatory cytokines, improved animal weight gain and metabolic profiling, and promoted locomotor recovery. Analysis of gut microbiome revealed that melatonin treatment decreased the Shannon index and reshaped the composition of intestinal microbiota. Melatonin-treated SCI animals showed decreased relative abundance of Clostridiales and increased relative abundance of Lactobacillales and Lactobacillus, which correlated with alteration of cytokine (monocyte chemotactic protein 1) expression and GI barrier permeability, as well as with locomotor recovery. Experimental induction of gut dysbiosis in mice before SCI (i.e., by oral delivery of broad-spectrum antibiotics) exacerbates neurological impairment after SCI, and melatonin treatment improves locomotor performance and intestinal integrity in antibiotic-treated SCI mice. The results suggest that melatonin treatment restores SCI-induced alteration in gut microbiota composition, which may underlie the ameliorated GI function and behavioral manifestations.