By measurement of intracellular free Ca2+ concentration using micro-fluorometric assay, whole cell calcium current and capacitance, regulative effects of extracelluar ATP on insulin secretion in primary rat β cells were studied. ATP elevated intracellular free Ca2+ concentration by mobilizing intercellular IP3 sensitive calcium pool, therefore it induced a strong secretion in resting β cells. Meanwhile, ATP attenuated depolarization-evoked Ca2+ influx and inhibited secretion induced by excitation of cell. The results demonstrated that ATP had dual regulative effects on insulin secretion in rat β cell. It is suggested extracellular ATP signal maybe have different effects on the two phases of insulin secretion in organisms: a negative feedback effect on the first phase, and a positive feedback effect on the second. Although, there are open questions to be resolved, the data support cue to the further study of mechanism underlying ATP regulation of insulin secretion.