Metabolic alterations in the somatosensory cortex play a crucial role in neuropathic pain development, as evidenced by magnetic resonance spectroscopy and mass spectrometry analyses of brain homogenates. However, investigating metabolic changes in specific neuronal subtypes during neuropathic pain development remains challenging. Here, utilizing a recently developed technique called single-cell mass spectrometry (SCMS), we investigated metabolomic alterations within excitatory glutamatergic neurons located in the primary somatosensory cortex (S1) during various stages of neuropathic pain. Specifically, we induced neuropathic pain in mice using a spared nerve injury (SNI) model and observed activation of glutamatergic neurons in layer II/III of S1 through c-Fos staining and electrophysiology. We profiled metabolic changes and performed pathway enrichment analysis in these neurons by SCMS during both acute and sub-chronic phases of SNI. Further analyses revealed metabolites whose alterations significantly correlated with changes in pain thresholds, as well as distinct temporal patterns of metabolite expression during pain progression. From these analyses, we identified several key metabolites (homogentisic acid, phosphatidylcholine, phosphorylcholine and rhein) and validated their causal roles in pain modulation via pharmacological interventions. Thus, our study provides a valuable resource for elucidating the neurometabolic regulatory mechanisms underlying neuropathic pain from a single-cell perspective.