We studied the effect of gap junctional coupling on the excitability of β-cells in slices of pancreas, which provide a normal environment for islet cells. The electrophysiological properties of β-cells from mice (C57Bl/6 background) lacking the gap junction protein connexin36 (Cx36^−/−) were compared with heterozygous (Cx36^+/−) and wild-type littermates (Cx36^+/+) and with frequently used wild-type NMRI mice. Most electrophysiological characteristics of β-cells were found to be unchanged after the knockout of Cx36, except the density of Ca²⁺ channels, which was increased in uncoupled cells. With closed ATP-sensitive K⁺ (K_ATP) channels, the electrically coupled β-cells of Cx36^+/+ and Cx36^+/− mice were hyperpolarized by the membrane potential of adjacent, inactive cells. Additionally, the hyperpolarization of one β-cell could attenuate or even stop the electrical activity of nearby coupled cells. In contrast, β-cells of Cx36^−/− littermates with blocked K_ATP channels rapidly depolarized and exhibited a continuous electrical activity. Absence of electrical coupling modified the electrophysiological properties of β-cells consistent with the reported increase in basal insulin release and altered the switch on/off response of β-cells during an acute drop of the glucose concentration. Our data indicate an important role for Cx36-gap junctions in modulating stimulation threshold and kinetics of insulin release.