Guanine-based purines are part of the purinergic system and recently have been shown to act as neuromodulators, interfering with acetylcholine release by enteric neurons in mouse colon. Due to the pivotal role played by enteric neurons in the control of gastrointestinal motility, the aim of the present study was to verify whether guanosine may affect gastric emptying and the mechanical tone, detected in vitro as changes in intraluminal pressure, of the isolated mouse stomach. Guanosine induced a TTX-insensitive concentration-dependent relaxation of isolated stomach, which at the maximal concentration tested (1 mM), reached about 60% of the relaxation induced by 1 mM isoproterenol. The inhibitory response was abolished by NBTI, a nucleoside uptake inhibitor. Moreover, guanosine-induced effects persisted in the presence of ODQ, an inhibitor of nitric oxide-dependent guanylate cyclase or TEA, a K⁺ channel antagonist, but they were progressively reduced by increasing concentrations of DDA, an adenylyl cyclase inhibitor. Oral guanosine decreased gastric emptying in a concentration-dependent manner. Results indicate that exogenous guanosine is able to inhibit gastric emptying and to induce gastric relaxation acting peripherally on the mouse stomach. The effect does not involve neural action potentials, nitric oxide release or opening K⁺ channels, but it dependents on guanosine cellular uptake and involves adenylyl cyclase activation.