In pancreatic β cells, ATP-sensitive potassium (K ATP ) channels are metabolic sensors that couple cell metabolism to electrical activity, and therefore K ATP channels regulate insulin secretion. We assume that down-regulating the expression of Kir6.2 subunits of K ATP channels may change calcium influx induced by glucose and insulin secretion regulated by K ATP channels. In our study, we employ Kir6.2-shRNA plasmid to down-regulate Kir6.2 expression in HIT-T15 cells. Then, we research the effect of down-regulation of Kir6.2 on K ATP current, cytoplasmic free Ca 2+ concentration and insulin secretion. All results illustrate that down-regulation of Kir6.2 subunits of K ATP channels in HIT-T15 cells affects K ATP current and insulin secretion, and fails to promote calcium influx. The results demonstrate the function of Kir6.2 subunits in electrophysiology characteristic, insulin secretion and calcium influx, and RNA interference provides a feasible alternative to study the function of Kir6.2 subunits in K ATP channels in different kinds of diabetes.