Polyphenol-rich foods, such as fruits and vegetables, are protective against cardiovascular diseases, but the mechanisms of the beneficial effects are still unknown. The goal of this research was to clarify actions of procyanidin trimer (C1) in rat aortic endothelial cells (RAECs). Procyanidin C1 at concentrations up to 50 μM was not cytotoxic to the RAECs. The addition of procyanidin C1 to RAECs exerted a time-dependent hyperpolarization measured using a membrane potential-dependent fluorescent probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol, whereas the hyperpolarization was significantly inhibited by the nonspecific K(+) channel inhibitor tetraethylammonium chloride (TEA). Moreover, procyanidin C1 elevated intracellular Ca(2+) influx, which was totally abolished in the presence of Ca(2+)-free solution with EGTA. Procyanidin C1 caused a significant increase in nitric oxide (NO) production. The effect was significantly inhibited by an NO synthase inhibitor, N(G)-monomethyl-l-arginine, or TEA. In conclusion, we demonstrated for the first time that procyanidin C1 plays a potent role in promoting Ca(2+)-mediated signals such as the hyperpolarization via multiple K(+) channel activations and the NO release in RAECs, suggesting that procyanidin C1 may represent novel and effective therapy for the treatment of cardiovascular diseases.