Some class I antiarrhythmic drugs induce a sporadic hypoglycemia by producing insulin secretion via inhibition of ATP-sensitive K⁺ (K_ATP) channels of pancreatic β-cells. It remains undetermined whether amiodarone produces insulin secretion by inhibiting K_ATP channels. In this study, effects of amiodarone on K_ATP channels, L-type Ca²⁺ channel, membrane potential, and insulin secretion were examined and compared with those of quinidine in a β-cell line (MIN6). Amiodarone as well as quinidine inhibited the openings of the K_ATP channel in a concentration-dependent manner without affecting its unitary amplitude in inside-out membrane patches of single MIN6 cells, and the IC₅₀ values were 0.24 and 4.9 μM, respectively. The L-type Ca²⁺ current was also inhibited by amiodarone as well as quinidine in a concentration-dependent manner. Although glibenclamide (0.1 μM) or quinidine (10 μM) significantly potentiated the insulin secretion from MIN6 cells, amiodarone (1 – 30 μM) failed to increase insulin secretion. Amiodarone (30 μM) and nifedipine (10 μM) significantly inhibited the increase in insulin secretion produced by 0.1 μM glibenclamide. Amiodarone (30 μM) produced a gradual decrease of the membrane potential, but did not produce repetitive electrical activity in MIN6 cells. Glibenclamide (1 μM) produced a slow depolarization, followed by spiking activity which was inhibited by 30 μM amiodarone. Thus, amiodarone is unlikely to produce hypoglycemia in spite of potent inhibitory action on K_ATP channels in insulin-secreting cells, possibly due to its Ca²⁺ channel–blocking action.