It is known that spinal cord injury (SCI) causes metabolic disorders, such as disrupted lipid and amino acid metabolism. However, the changes of metabolites in the spinal cord remain elusive. In the present study, adult Sprague-Dawley rats underwent spinal cord transection surgery. Ambient air flow-assisted desorption electrospray ionization mass spectrometry imaging was performed to identify the localization of metabolites in the white and gray matter and the ventral and dorsal horns within the spinal cord sections. The results revealed that 42 metabolites were specifically increased in the gray matter, and 90 metabolites were increased in the white matter compared with that in the sham group. In the ventral and dorsal horns, 86 and 103 metabolites, respectively, exhibited specific increases after injury. By contrast, numerous metabolites, especially lipids, were significantly decreased after SCI. Phosphatidylserine and phosphatidylethanolamine were mainly decreased in the gray matter, while cholesterol and ceramide were mainly decreased in the white matter. Specifically, phosphatidylethanolamine was detected at low levels in the dorsal horn following injury. However, the phosphatidylserine level decreased in the ventral horn. The functional enrichment of these metabolites via Kyoto Encyclopedia of Genes and Genomes analysis further confirmed the profile differences in the white and gray matter, as well as in the ventral and dorsal horns after SCI. These results provide valuable information on the metabolite profiles across various anatomical regions of the spinal cord following SCI, which may support the development of precise treatment strategies for patients.