Obestatin is a hormone released from the stomach deriving from the same peptide precursor as ghrelin. It is known to act as an anorectic hormone decreasing food intake but contrasting results have been reported about the effects of obestatin on gastrointestinal motility. The aim of the present study was to investigate whether this peptide may act on the gastric longitudinal smooth muscle by using a combined mechanical and electrophysiological approach. Gastric fundal strips from mice were mounted in organ baths for isometric recording of the mechanical activity. Obestatin caused a TTX-insensitive decrease of the basal tension and a reduction in amplitude of the neurally-induced cholinergic contractile responses even in the presence of the nitric oxide synthesis inhibitor L-N-nitro-arginine. Obestatin reduced the amplitude of the response to the ganglionic stimulating agent dimethylphenyl piperazinium iodide (DMPP) but did not influence that to methacholine. In non-adrenergic, non-cholinergic conditions, obestatin still decreased the basal tension of the preparations without influencing the neurally-induced relaxant responses. Electrophysiological experiments, performed by a single microelectrode inserted in a gastric longitudinal smooth muscle cell, showed that obestatin increased the membrane resistance, caused the inhibition of Ca²⁺ currents and positively shifted their voltage threshold of activation. In conclusion, the present results indicate that obestatin, other than exerting a neuromodulatory action at the postganglionic level, directly influences the gastric longitudinal smooth muscle. The reduction of the contractile responses might contribute to the distension of the gastric wall, one of the major signal involved in the regulation of food intake.