Interfaces are crucial for the water management in polymer electrolyte membrane fuel cells (PEMFCs). The introduction of a microporous layer (MPL) had a revolutionary effect on the water distribution by improving the interface between the catalyst layer and the gas diffusion layer substrate (GDL-S). Hence, it is vital to maximize the improvement by further characterizing and advancing the properties of the interfaces, in this case the MPL/GDL-S interface. This study aims at fabricating a GDL with an MPL that intrudes into the GDL-S, analyzing the impact on the GDL-S structure and on PEMFC performance. Mercury intrusion porosimetry (MIP) and ex situ X-ray tomography (XTM) show that the intrusion of the MPL into the hydrophobic GDL-S proceeds via the preferential filling of the GDL-S macropores, thereby reducing their size and volume fraction in the GDL-S. While an intruding MPL leads to a small performance increase under wet PEMFC operating conditions, this improvement could only be achieved by a careful management between the extent of MPL intrusion and the partial macropore blocking in the GDL-S. Furthermore, the impact of MPL intrusion on the liquid water saturation of the GDL was quantified by operando XTM. The results provide design guidelines for improved GDLs.