Src Homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1) interacts specifically with GluN2A subunit of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors in spinal cord dorsal horn. This molecular interaction is involved in the development of GluN2A-dependent spinal sensitization of nociceptive behaviors. Intrathecal application of a GluN2A-derived polypeptide (short for pep-GluN2A) has been shown to disturb spinal GluN2A/SHP1 interaction and inhibit inflammatory pain. Here we found that SHP1 was also located at dorsal root ganglion (DRG) neurons and formed complexes with GluN2A subunit. Peripheral inflammation activated SHP1 in DRG neurons, which promoted GluN2A tyrosine phosphorylation. The SHP1 binding to GluN2A facilitated the glutamate release from primary afferent fibers and exaggerated nociceptive synaptic transmission onto postsynaptic spinal cord neurons. Our data showed that intradermal application of pep-GluN2A disrupted GluN2A/SHP1 interaction in DRG neurons, attenuated the ability of GluN2A subunit-containing NMDA receptors to regulate the presynaptic glutamate release and more importantly, alleviated the pain hypersensitivity caused by carrageenan, complete Freund's adjuvant and formalin. The neuropathic pain induced by spared nerve injury was also ameliorated by intradermal pep-GluN2A application. These data suggested that disruption of GluN2A/SHP1 interaction in DRG neurons generated an effective analgesic action against pathological pain.