Cyclic AMP (cAMP) and Ca²⁺ are key regulators of exocytosis in many cells, including insulin-secreting β cells. Glucose-stimulated insulin secretion from β cells is pulsatile and involves oscillations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), but little is known about the detailed kinetics of cAMP signaling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the submembrane space of single MIN6 cells and primary mouse β cells. These oscillations were preceded and enhanced by elevations of [Ca²⁺]_i. However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca²⁺]_i rise, indicating that adenylyl cyclase activity may be controlled also by the substrate concentration. The cAMP oscillations correlated with pulsatile insulin release. Whereas elevation of cAMP enhanced secretion, inhibition of adenylyl cyclases suppressed both cAMP oscillations and pulsatile insulin release. We conclude that cell metabolism directly controls cAMP and that glucose-induced cAMP oscillations regulate the magnitude and kinetics of insulin exocytosis.