The pancreatic islets of Langerhans are highly vascularized micro-organs that play a key role in the regulation of blood glucose homeostasis. The specific arrangement of endocrine cell types in islets suggests a coupling between morphology and function within the islet. Here, we established a line-scanning confocal microscopy approach to examine the relationship between blood flow and islet cell type arrangement by real-time in vivo imaging of intra-islet blood flow in mice. These data were used to reconstruct the in vivo 3D architecture of the islet and time-resolved blood flow patterns throughout the islet vascular bed. The results revealed 2 predominant blood flow patterns in mouse islets: inner-to-outer, in which blood perfuses the core of β cells before the islet perimeter of non–β cells, and top-to-bottom, in which blood perfuses the islet from one side to the other regardless of cell type. Our approach included both millisecond temporal resolution and submicron spatial resolution, allowing for real-time imaging of islet blood flow within the living mouse, which has not to our knowledge been attainable by other methods.