BACKGROUND: Neuropathic pain is a complex condition resulting from damage or disease in the somatosensory nervous system, causing significant physical and emotional distress. Despite its profound impact, the underlying causes and treatment methods of neuropathic pain remain poorly understood. METHODS: To better understand this condition, we conducted the first study examining the spatial distribution and dynamic expression changes of N-glycan molecules that play a crucial role in nervous system function and sustainable pain signal transmission across multiple regions of the spinal cord and brain in an experimentally induced neuropathic pain model, using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). RESULTS: Our findings revealed that neuropathic pain induces dynamic changes in N-glycan expression across various regions of the spinal cord and brain. Notably, we discovered distinct glycan profiles between the spinal cord and brain, with N-glycans downregulated in the spinal cord and upregulated in the brain at a time when mechanical allodynia is sustained following spinal nerve ligation (SNL). Significant changes in N-glycan expression were observed in the dorsal laminae IV/V/VI and the ventral horn of the spinal cord. Additionally, marked changes were detected in the contralateral regions of the primary sensory cortex (S1) and the primary sensory cortex hindlimb area (S1HL). Furthermore, we observed significant upregulation of N-glycan expression in the thalamus, anterior cingulate cortex (ACC), and medial prefrontal cortex (mPFC) in both ipsilateral and contralateral regions of the brain. CONCLUSIONS: Given that N-glycans are implicated in pain processing yet their precise role remains unclear, our study highlights the need to explore N-glycosylation with a more nuanced focus on both the spinal cord and brain. This research provides new insights into the mechanisms of persistent neuropathic pain and lays the groundwork for future studies and the development of targeted therapeutic strategies.