Objective To characterize the three-dimensional (3D) course of the mouse proximal tubules. Methods Three C57/BL/6J mice were fixed by perfusion and embedded in Epon 812. Tissue blocks were cut perpendicular to the longitudinal axis of the kidney, then a total of 1 200, 2.5-μm-thick consecutive sections were obtained from the surface to the outer stripe of the outer medulla. After image recordings and alignment, proximal tubules from 58 nephrons were traced and 3D-reconstructed with a series of computer programs written in C language. Results In cortical labyrinth, all of the proximal tubules left their glomeruli ascending toward the renal surface for a distance of 100-1 400μm, then returned and made coils around their own glomeruli and occupied separate domains which did not intermingle with the neighboring nephron tubules. The proximal tubules originating from the superficial nephrons and midcortical nephrons formed tight clusters and occupied smaller volume than the proximal tubules originating from the juxtamedullary nephrons. In medullary rays, the pars recta were arranged in a specific pattern: proximal tubules originating in the superficial cortex were arranged in the center, whereas proximal tubules originating deeper in the cortex became layered more peripherally, and in the deepest juxtamedullary nephrons, it was impossible to define a pars recta since the “straight” part did not run straightly in the outer stripe of the outer medulla. All the proximal tubules terminated at the boundary between the outer stripe of the outer medulla and the inner stripe of the outer medulla and transitioned to descending thin limb of the Henle’s loop. Conclusion In the cortical labyrinth and medullary rays, the courses of the initial, convoluted and straight proximal tubules run in different regions and in different biological environment, which supply implications for evaluting segment distributio