Grape seed is an important natural bioactive product with various health benefits. Interstitial cells of Cajal (ICCs) are pacemaker cells in the gastrointestinal (GI) tract. The present study investigated the effects of grape seed powder (GSP) on ICC properties and GI motility. GSP depolarized the pacemaker potentials of ICCs in a dose‑dependent manner. Y25130 or SB269970 slightly inhibited GSP‑induced effects. However, Y25130 and SB269970 together completely blocked GSP-induced effects. In the presence of inhibitors of protein kinase C, protein kinase A, or mitogen-activated protein kinase, GSP‑induced ICC depolarization was inhibited. GSP increased the intestinal transit rate in normal mice and in mice with acetic acid-induced GI motility disorder. In addition, the levels of motilin and substance P were elevated after GSP dosing. These results demonstrate that GSP can regulate GI motility, and therefore, it is a potential therapeutic agent for treating GI motility disorders.