Abstract Experimental evidence in animal models suggests that adenosine is involved in the regulation of digestive functions. This study examines the influence of adenosine on the contractile activity of human colon. Reverse transcription‐polymerase chain reaction revealed A1and A2areceptor expression in colonic neuromuscular layers. Circular muscle preparations were connected to isotonic transducers to determine the effects of 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX; A1receptor antagonist), ZM 241385 (A2areceptor antagonist), CCPA (A1receptor agonist) and 2‐[(p‐2‐carboxyethyl)‐phenethylamino]‐5′‐N‐ethyl‐carboxamide‐adenosine (CGS 21680; A2areceptor agonist) on motor responses evoked by electrical stimulation or carbachol. Electrically evoked contractions were enhanced by DPCPX and ZM 241385, and reduced by CCPA and CGS 21680. Similar effects were observed when colonic preparations were incubated with guanethidine (noradrenergic blocker), L‐732,138, GR‐159897 and SB‐218795 (NK receptor antagonists). However, in the presence of guanethidine, NK receptor antagonists and Nω‐propyl‐l‐arginine (NPA; neuronal nitric oxide synthase inhibitor), the effects of DPCPX and CCPA were still evident, while those of ZM 241385 and CGS 21680 no longer occurred. Carbachol‐induced contractions were unaffected by A2areceptor ligands, but they were enhanced or reduced by DPCPX and CCPA, respectively. When colonic preparations were incubated with guanethidine, NK antagonists and atropine, electrically induced relaxations were partly reduced by ZM 241385 or NPA, but unaffected by DPCPX. Dipyridamole or application of exogenous adenosine reduced electrically and carbachol‐evoked contractions, whereas adenosine deaminase enhanced such motor responses. In conclusion, adenosine exerts an inhibitory control on human colonic motility. A1receptors mediate direct modulating actions on smooth muscle, whereas A2areceptors operate through inhibitory nitrergic nerve pathways.