Glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells is critical to the maintenance of blood glucose homeostasis in animals. Both decrease in pancreatic beta-cell mass and defects in beta-cell function contribute to the onset of diabetes, although the underlying mechanisms remain largely unknown. Molecular imaging techniques can help beta-cell study in a number of ways. High-resolution fluorescence imaging techniques provide novel insights into the fundamental mechanisms underlying GSIS in isolated beta-cells or in situ in pancreatic islets, and dynamic changes of beta-cell mass and function can be noninvasively monitored in vivo by imaging techniques such as positron emission tomography and single-photon emission computed tomography. All these techniques will contribute to the better understanding of the progression of diabetes and the search for the optimized therapeutic measures that reverse deficits in beta-cell mass and function.