Transport properties, performance, and durability of a proton exchange fuel cell (PEMFC) highly depend on microstructure and spatial distribution of components in the gas diffusion layer (GDL), microporous layer (MPL), and catalyst layers (CLs) of the fuel cell. Modeling of transport properties and understanding of these effects are challenging due to limited understanding of actual three-dimensional (3D) structure of the components, especially over a wide range of length scales. In this work, 3D imaging on multiple scales, namely electron tomography on a nanoscale, focused ion beam–scanning electron microscopy on a microscale, and 3D X-ray microscopy on a macroscale, was applied to obtain 3D reconstructions of the actual CL, MPL, and GDL microstructure. Direct numerical simulations on 3D data sets with an upscaling approach were applied to demonstrate the capability to simulate overall electrical conductivity of the system. Details of the process, challenges, and results are described.