Virus removal filters are designed to ensure very high levels of removal of viruses and virus-like particles during bioprocessing, but the performance of some virus removal filters can be compromised at high-throughputs, after process disruptions, or during operation at low pressures. Several studies have hypothesized that the different behavior is due to differences in underlying pore morphology, but current techniques are limited to examining the 2D pore structure. Here, we use the combination of a focused ion beam and scanning electron microscopy (FIB-SEM) to obtain 3D reconstructions of the pore structure of the asymmetric Viresolve® Pro virus removal filter. Images were obtained through a 3 μm section into the membrane starting at the size-selective skin. The membrane porosity decreases from 41 to 17% as one approaches the filter exit. Model simulations based on flow through the full 3D pore reconstruction show 100% virus retention, with all virus particles captured at least 400 nm from the filter exit. A pore-network model was developed from the reconstructions and used to evaluate the body and throat size distribution and pore interconnectivity. The number of throats is approximately twice the number of bodies, with an average throat size of 21 nm within the selective skin. The pore interconnectivity remains relatively constant at a value of 4 throats per body. These results provide insights into the underlying pore structure of the Viresolve® Pro virus removal filter as well as a general framework for characterizing the 3D pore space in nanoporous membranes.