The structural organisation of pancreatic β-cells in the islets of Langerhans is relatively unknown. Here, using three-dimensional (3D) two-photon, 3D confocal and 3D block-face serial electron microscopy, we demonstrate a consistent in situ polarisation of β-cells and define three distinct cell surface domains. An apical domain located at the vascular apogee of β-cells, defined by the location of PAR-3 (also known as PARD3) and ZO-1 (also known as TJP1), delineates an extracellular space into which adjacent β-cells project their primary cilia. A separate lateral domain, is enriched in scribble and Dlg, and colocalises with E-cadherin and GLUT2 (also known as SLC2A2). Finally, a distinct basal domain, where the β-cells contact the islet vasculature, is enriched in synaptic scaffold proteins such as liprin. This 3D analysis of β-cells within intact islets, and the definition of distinct domains, provides new insights into understanding β-cell structure and function.