Tong-Xie-Yao-Fang (TXYF) is a prescription in traditional chinese medicine (TCM), used for relieving abdominal pain associated with irritable bowel syndrome. The aim of the present study was to investigate the effects and mechanism of TXYF on experimental visceral hypersensitivity (VH) models. TXYF affected the abdominal withdrawal reflex produced by colonic distention in maternal separation-induced visceral hypersensitivity rats, in a dosage-dependent manner. TXYF significantly decreased serotonin (5-HT) levels in serum and corticotrophin releasing factor (CRF) concentrations in the brain. Moreover, it was found that VH alleviation by TXYF was dependent on the substance P (SP) expression in the colon mucosa. These results suggest that TXYF attenuates behavioral hyperalgesia by regulating substance associated with the brain-gut axis, including decreasing the expression of 5-HT and SP in the periphery and that of CRF in the center.