Insulin secretion from pancreatic β cells in response to high glucose (HG) critically depends on the inhibition of K_ATP channel activity in HG. It is generally believed that HG-induced effects are mediated by the increase in intracellular ATP, but here, we showed that, in INS-1 cells, endocytosis of K_ATP channel plays a major role. Upon HG stimulation, resting membrane potential depolarized by 30.6 mV (from −69.2 to −38.6 mV) and K_ATP conductance decreased by 91% (from 0.243 to 0.022 nS/pF), whereas intracellular ATP was increased by only 47%. HG stimulation induced internalization of K_ATP channels, causing a significant decrease in surface channel density, and this decrease was completely abolished by inhibiting endocytosis using dynasore, a dynamin inhibitor, or a PKC inhibitor. These drugs profoundly inhibited HG-induced depolarization. Our results suggest that the control of K_ATP channel surface density plays a greater role than ATP-dependent gating in regulating β cell excitability.