Background/Aims: Although neurotensin (NT) stimulates colon motility and the passage of intestinal contents, the associated mechanism of action remains unclear. The objective of this study was to investigate the effects of NT on colon motility using isolated rat colon. Methods: Intraluminal pressure was measured at both the proximal and distal portions of the isolated colon. An isolated rat colon was perfused with Krebs solution via the superior mesenteric artery. After stabilization, NT was administered in concentrations of 14, 28, 138 and 276 pM. After pretreatment with phentolamine, propranolol, hexamethonium, atropine or tetrodotoxin, NT was administered at a concentration of 276 pM, and then the intraluminal pressure was monitored. Results: NT significantly increased colon motility at concentrations of 14, 28, 138, and 276 in the proximal colon (25.1±6.5%, 175.4±117.0%, 240.8±115.1% and 252.3±110.6%, respectively) and in the distal colon (35.6±11.8%, 97.5±35.1%, 132.7±36.7% and 212.1±75.2%, respectively). The stimulant effect of NT was more potent in the proximal colon, in a concentration-dependent manner (P<0.05). The stimulant effect of NT was significantly inhibited by atropine at both the proximal and distal colon and by tetrodotoxin at the proximal colon, but not by tetrodotoxin at the distal colon and not by propranolol, phentolamine, or hexamethonium at both the proximal and distal colon. Conclusions: NT increased colon motility at both the proximal and distal portions of the rat colon. The effects were more prominent at the proximal portion. The results of this study suggest that the stimulant action of NT may be mediated by local cholinergic muscarinic receptors.