In this study, we examined the effects of serotonin (5‐HT)₃ receptor antagonists (5‐HT₃RAs) including ramosetron, alosetron, and cilansetron on colonic nociceptive threshold in rats. Furthermore, we established a restraint stress‐induced colonic hyperalgesia model in rats, and compared the inhibitory effects of 5‐HT₃RAs on restraint stress‐induced colonic hyperalgesia and diarrhoea with those of loperamide, trimebutine, tiquizium and polycarbophil. The colonic nociceptive threshold was measured as the balloon pressure at the time the rat showed a nociceptive response during colonic distension by an intrarectally inserted balloon. Oral administration of ramosetron (3–30 μg kg⁻¹), alosetron (30–300 μg kg⁻¹), or cilansetron (30–300 μg kg⁻¹) increased the colonic nociceptive threshold in a dose‐dependent manner in non‐stressed rats. Restraint stress for 1 h significantly decreased the colonic nociceptive threshold, but ramosetron (0.3–3 μg kg⁻¹), alosetron (3–30 μg kg⁻¹), cilansetron (3–30 μg kg⁻¹) and trimebutine (100–1000 mg kg⁻¹) significantly inhibited the decrease in the threshold. Loperamide (3–30 mg kg⁻¹), tiquizium (100–1000 mg kg⁻¹) and polycarbophil (1000 mg kg⁻¹) did not affect the restraint stress‐induced decrease in the colonic nociceptive threshold. All drugs tested in this study showed dose‐dependent inhibition of restraint stress‐induced diarrhoea in rats. These results indicate that, unlike existing antidiarrhoeal and spasmolytic agents, 5‐HT₃RAs have inhibitory effects on colonic nociception, and prevented restraint stress‐induced both diarrhoea and hyperalgesia at almost the same doses in rats. This suggests that the 5‐HT₃RAs may be useful in ameliorating both colonic hyperalgesia and diarrhoea in patients with irritable bowel syndrome.