Glucose stimulates insulin secretion (IS) by raising Ca²⁺ and cAMP in pancreatic b-cells. Whereas Ca²⁺ is the principal trigger, cAMP is an important amplifier of IS. Insulin is stored in secretory vesicles as a crystalline zinc complex. In this study we tested the hypothesis that Zn²⁺ co-released with insulin exerts negative feedback on cAMP signaling and IS from glucose-stimulated b-cells. The sub-plasma membrane concentrations of Zn²⁺, Ca²⁺ and cAMP were measured in single MIN6 cells using evanescent wave microscopy and FluoZin-3, Fura Red and a fluorescent protein translocation sensor, respectively. A similar translocation assay for the plasma membrane lipid PIP₃ was used as read-out of IS. Glucose stimulation of MIN6 cells induced pronounced Ca²⁺, cAMP and PIP₃ oscillations reflecting pulsatile IS, but sometimes these responses faded with time. Glucose also elevated the sub-membrane Zn²⁺ concentration, and some cells showed synchronized oscillations of Zn²⁺ and Ca²⁺. Hyperpolarization or blocking of voltage- dependent Ca²⁺ channels prevented both Ca²⁺ and Zn²⁺ oscillations. Addition of 30 uM exogenous zinc caused an increase of sub-membrane Zn²⁺, a marked inhibition of the cAMP and PIP₃ responses to glucose as well as suppression of IS detected with ELISA. Whereas the inhibitory effects of added zinc remained after omission of the cation and chelation of residual extracellular zinc with Ca-EDTA, the cell-permeable heavy metal chelator TPEN readily restored cAMP oscillations and IS. TPEN amplified the cAMP and PIP₃ oscillations also in the absence of added zinc and immediately rescued the spontaneously faded responses. These data indicate that Zn²⁺ co- released with insulin feedback-inhibits intracellular cAMP signaling and suppresses IS.