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