Previous studies demonstrated the efficacy of electro-acupuncture (EA) in relieving chronic visceral hypersensitivity (CVH) in IBS rats. However, ST25 which is a key acupoint for patients with IBS has not been reported in these experiments. Eight CVH rats were treated by EA at both ST25 and ST37 for 20 min, once daily for seven consecutive days, model rats (n = 8) and normal rats (n = 8) as controls. After the first EA treatment, the abdominal withdrawal reflex scores were investigated to evaluate the pain threshold. After seven EA treatments, the concentrations of 5-hydroxytryptamine (5-HT), 5-HT3 receptor (5-HT3R) and 5-HT4 receptor (5-HT4R) in colon tissue were assayed quantitatively by enzyme-linked immunosorbent assay. The results showed that EA improved the pain threshold of CVH rats, reduced the 5-HT concentration and increased the 5-HT4R concentration, but had no effect on the 5-HT3R concentration. Further studies are needed to optimize the choice of two-matching points for EA in the treatment of CVH rats.