A critical role of circadian oscillators in orchestrating insulin secretion and islet gene transcription has been demonstrated recently in whole islets. We performed a parallel analysis of the molecular properties of α-cell and β-cell oscillators using a mouse model expressing three reporter genes: one labeling α cells, one specific for β cells, and a third monitoring circadian gene expression. These experiments showed that α-cellular and β-cellular clocks are oscillating with distinct phases in vivo and in vitro. Diurnal transcriptome analysis in separated α and β cells revealed that a high number of genes, with key roles in islet physiology, are differentially expressed in these cell types. Moreover, temporal insulin and glucagon secretion exhibited distinct oscillatory profiles both in vivo and in vitro. Altogether, our data indicate that differential entrainment characteristics of circadian α-cell and β-cell clocks are an important feature in the temporal coordination of endocrine function and gene expression.