Abstract The functional role of serotonergic 5-HT1A receptors in the modulation of visceral pain is controversial. The objective of this study was to systematically examine the mechanism and site of action of a selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (DPAT) on visceral pain. In the behavioral model of visceral pain, systemic injection (5–250μg/kg) of DPAT produced a significant increase in the viscero-motor response (VMR) to colorectal distension (CRD) and this effect was blocked by the selective 5-HT1A receptor antagonist WAY-100135 (5mg/kg, s.c.). Similarly, intrathecal (i.t.) injection (5μmol) of DPAT into the lumbo-sacral (L6–S1) spinal cord produced a significant increase in VMR. The administration of N-methyl d-aspartate (NMDA) receptor antagonist AP5 (50μg/kg) prior to DPAT injection completely blocked the pronociceptive effect of DPAT. Similarly, DPAT failed to increase VMR in rats chronically treated with NR1 subunit-targeted antisense oligonucleotide (ON), whereas the drug increased VMR in rats treated with mismatched-ON. Chronic i.t. injection of allylglycine (AG), a γ-amino decarboxylase (GAD) enzyme inhibitor, produced significant increase in VMRs, suggesting that the inhibition of GABA synthesis produces pronociception. In AG-treated rats, i.t. injection of DPAT failed to further increase in VMR, suggesting that the DPAT action is linked to GABA release. Similarly, WAY-100135 failed to attenuate VMR in AG-treated rats, suggesting that unlike DPAT, AG action is not via the activation of 5-HT1A receptors. In electrophysiology experiments, DPAT (50μg/kg) significantly increased the responses of spinal neurons to CRD, but did not influence the mechanotransduction property of CRD-sensitive pelvic nerve afferent fibers. The effect of DPAT on spinal neurons remained unaffected when tested in spinal-transected (C1–C2) rats. These results indicate that the 5-HT1A receptor agonist DPAT produces pronociceptive effects, primarily via the activation of presynaptic 5-HT1A receptors in GABAergic neuron to restrict GABA release and thereby disinhibits the excitatory glutamatergic neurons in the spinal cord.