Homeostasis of blood glucose is mainly regulated by the coordinated secretion of glucagon and insulin from α- and β-cells within the islets of Langerhans. The release of both hormones is Ca²⁺ dependent. In the current study, we used confocal microscopy and immunocytochemistry to unequivocally characterize the glucose-induced Ca²⁺ signals in α- and β-cells within intact human islets. Extracellular glucose stimulation induced an opposite response in these two cell types. Although the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in β-cells remained stable at low glucose concentrations, α-cells exhibited an oscillatory [Ca²⁺]_i response. Conversely, the elevation of extracellular glucose elicited an oscillatory [Ca²⁺]_i pattern in β-cells but inhibited low-glucose–induced [Ca²⁺]_i signals in α-cells. These Ca²⁺ signals were synchronic among β-cells grouped in clusters within the islet, although they were not coordinated among the whole β-cell population. The response of α-cells was totally asynchronic. Therefore, both the α- and β-cell populations within human islets did not work as a syncitium in response to glucose. A deeper knowledge of α- and β-cell behavior within intact human islets is important to better understand the physiology of the human endocrine pancreas and may be useful to select high-quality islets for transplantation.