Recently, the role of EphB receptor (EphBR) tyrosine kinase and their ephrinB ligands in spinal pain-related neural plasticity has been identified. To test whether Src-family non-receptor tyrosine kinase-dependent glutamatergic N-methyl-D-aspartate receptor (NMDAR) NR2B subunit phosphorylation underlies lumbosacral spinal EphBR activation to mediate cross-organ sensitization between the colon and the urethra, external urethra sphincter electromyogram (EUSE) activity evoked by pelvic nerve stimulation and protein expression at the lumbosacral (L6-S2) dorsal horn were studied before and after intra-colonic mustard oil (MO) instillation. We found MO instillation produced colon-urethra reflex sensitization in accompaniment with an up-regulation of endogenous ephrinB2 expression as well as phosphorylation of EphB1/2, Src-family kinase and NR2B tyrosine residues. Intrathecal immunoglobulin fusion protein of EphB1 and EphB2 as well as PP2 reversed the reflex sensitization and NR2B phosphorylation caused by MO. All these results suggest EphBR-ephrinB interactions, which provoke Src-family kinases-dependent NMDAR NR2B phosphorylation at the lumbosacral spinal cord level is involved in cross-organ sensitization contributing to the development of viscero-visceral refer pain between the bowel and the urethra.