We have previously shown that NO has stimulatory and inhibitory effects on insulin secretion at low and high concentrations, respectively. The present study investigated effects of NO on K_ATP channels of rat β cells by patch clamp analysis to elucidate the mechanism for the dual effect. NOC7 at 0.5 μM suppressed K_ATP channels activated by diazoxide in the cell-attached and perforated whole-cell modes but failed to suppress them in the inside-out mode. The inhibitory effect in the cell-attached mode was abolished by the soluble guanylate cyclase inhibitor ODQ and by the protein kinase G inhibitor KT5823. Moreover, 0.5 μM NOC7 failed to suppress the channel activity in the presence of the mitochondrial uncoupler FCCP. In contrast, 10 μM NOC7 activated K_ATP channels in the cell-attached and perforated whole-cell modes, although it had no effect on the channels in the inside-out mode. The K_ATP currents evoked by 10 μM NOC7 in the cell-attached mode were not inhibited by ODQ. The dual effect of NOC7 at 0.5 and 10 μM was observed in the same patch. Taken together, these results suggest that low-concentration NO exerts an inhibitory effect on K_ATP channels of β cells, which is induced through the cGMP/protein kinase G pathway, whereas high-concentration NO activates K_ATP channels through the mechanism independent of cGMP.