Cell-to-cell interactions play an important role in insulin secretion. Compared to intact islets, dispersed pancreatic beta cells show increased basal and decreased glucose-stimulated insulin secretion. In this study, we used mouse MIN6B1 cells to investigate the mechanisms that control insulin secretion when cells are in contact with each other or not. RNAi-mediated silencing of the adhesion molecule E-cadherin in confluent cells reduced glucose stimulated secretion to the levels observed in isolated cells, but had no impact on basal secretion. Dispersed cells presented high cytosolic Ca²⁺ activity, depolymerized cytoskeleton and ERK1/2 activation in low glucose conditions. Both the increased basal secretion and the spontaneous Ca²⁺ activity were corrected by transient removal of Ca²⁺ or by prolonged incubation of cells in low glucose, a procedure that restored the ability of dispersed cells to respond to glucose (11-fold stimulation). In conclusion we show that dispersed pancreatic beta cells can respond robustly to glucose once their elevated basal secretion has been corrected. The increased basal insulin secretion of dispersed cells is due to spontaneous Ca²⁺ transients that activate downstream Ca²⁺-effectors, while engagement of cell adhesion molecules including E-cadherin contributes to the greater secretory response to glucose seen in cells with normal intercellular contacts.