Glucagon-like peptide-1 (GLP-1) analogue ROSE-010 can provide effective pain relief from irritable bowel syndrome (IBS). However, the underlying biological mechanism is still unknown. Here, we investigate the effect of GLP-1 analogue exendin-4 on visceral hypersensitivity in colonic sensitized rats. Rat models of visceral hypersensitivity were established by intra-colonic infusion of acetic acid in 10-day-old Sprague-Dawley rats. Visceral sensitivity was assessed by measurement of abdominal withdrawal reflex (AWR) and electromyography (EMG). Exendin-4 with doses of 1, 5, and 10 μg/kg were intraperitoneally administered, respectively. The expressions of serotonin transporter (SERT) and tryptophan hydroxylase-1 (TPH-1) in colonic tissues were detected by RT-PCR and Western blot, respectively. The levels of serotonin (5-HT) and GLP-1 were measured by ELISA assay. Visceral hypersensitivity after neonatal colonic sensitization was verified. The colonic sensitized rats showed low levels of GLP-1 in plasma and high levels of 5-HT in plasma and colonic tissue (P<0.05). Exendin-4 dose-dependently reduced visceral hypersensitivity in colonic sensitized rats. The AWR scores in colonic sensitized rats with exendin-4 (5 μg/kg) reduced to 1.56±0.53 (P=0.013 vs. 2.33±0.50), 2.23±0.45 (P=0.008 vs. 3.0±0.5) during CRD at 40, and 60 mmHg, respectively. Similar findings were showed at dose of 10 μg/kg. Exendin-4 (5 μg/kg and 10 μg/kg) reduced the EMG during CRD at 40, 60, 80 mmHg (P<0.01). Exendin-4 (5.0 μg/kg or 10.0 μg/kg) significantly decreased the 5-HT colonic levels (2.343±0.447, 2.175±0.360 ng/100 mg vs. 3.607±0.628 ng/100 mg, P<0.05). The SERT protein expressions in colonic tissues in colonic sensitized rats were significantly increased with exendin-4 at doses of 1, 5 or 10 μg/kg (0.759±0.068, 0.942±0.037, 0.944±0.097 vs. 0.552±0.047, P<0.05, respectively), and the SERT mRNA expression also increased after treatment with exendin-4. The colonic sensitized rats showed lower TPH-1 levels after treatment with exendin-4 (P<0.05). These findings suggest that exendin-4 reduce visceral hypersensitivity and this may be associated with up-regulating SERT expression, and down-regulating TPH-1 expression.