AIMS: Sustained visceral hypersensitivity is a hallmark of irritable bowel syndrome (IBS) could be partially explained by enteric neural remodeling. Particularly, synaptic plasticity in the enteric nervous system, a form of enteric "memory", has been speculated as a participant in the pain maintenance in IBS. This study aimed to elucidate the role of ephrinB2/ephB2 in enteric synaptic plasticity and visceral pain in IBS. MATERIALS AND METHODS: EphrinB2/ephB2 expression and synaptic plasticity were assessed in colonic tissues from IBS patients, and rats induced by Trichinella spiralis infection and those treated with ephB2-Fc (an ephB2 receptor blocker) or ifenprodil (a selective NR2B antagonist). Furthermore, abdominal withdrawal reflex scores to colorectal distention and mesenteric afferent firing were assessed. EphrinB2-Fc(an ephB2 receptor activator) induced enteric synaptic plasticity was further evaluated in longitudinal muscle-myenteric plexus(LMMP) cultures and primary cultured myenteric neurons. KEY FINDINGS: EphrinB2/ephB2 was specifically expressed in colonic nerves and upregulated in IBS patients and rats, which was correlated with pain severity. The functional synaptic plasticity, visceral sensitivity to colorectal distention and colonic mesenteric afferent activity to mechanical and chemical stimulus were enhanced in IBS rats, and were blocked by ephB2-Fc or ifenprodil treatment. EphrinB2-Fc promoted the phosphorylation of NR2B in IBS rats and LMMP cultures, and could mediate sustained neural activation via increased [Ca] and raised expression of synaptic plasticity-related early immediate genes, including c-fos and arc. SIGNIFICANCE: EphrinB2/ephB2 facilitated NR2B-mediated synaptic potentiation in the enteric nervous system that may be a novel explanation and potential therapeutic target for sustained pain hypersensitivity in IBS.