Sustained pain hypersensitivity is the hallmark of stressed colon which could be partially explained by central sensitization with synaptic plasticity, the key mechanism of memory. We previously identified that synaptic plasticity of enteric nerve system (ENS) contributed to peripheral pain maintaining in the gut. However, the mechanisms of enteric "memory" formation remain elusive. In this study, rats were exposed to water avoidance stress (WAS) or sham stress (SS), with cromolyn sodium or physiological saline injected intraperitoneally 30minutes before stress every day. The abdominal withdrawal reflex scores, mesenteric afferent nerve activity, enteric neural c-fos expression, and enteric synaptic plasticity were assessed, and mast cell infiltration and degranulation. Furthermore, colonic mucosal mediators-induced enteric synaptic plasticity and the role of mast cell-derived nerve growth factor (NGF), tryptase, and histamine were investigated via ex vivo longitudinal muscle-myenteric plexus (LMMP) organotypic culture. It is shown that mast cell stabilizing inhibited WAS-induced visceral hypersensitivity through enhancing visceral pain threshold, decreasing spontaneous and distention-induced mesenteric afferent firing, and downregulating enteric neural activation (c-fos). Importantly, WAS led to evident enteric synaptic plasticity, but decreased by cromolyn. Water avoidance stress-derived mucosal supernatants markedly enhanced the c-fos expression and enteric synaptic plasticity in LMMP tissues, which could be eliminated by mast cell inhibition or NGF neutralization, but not tryptase or histamine blocking. In conclusion, mast cells/NGF pathway may be the key regulator of synaptic plasticity of ENS and participate in the formation of chronic stress-induced sustained visceral hypersensitivity.