A series of mixed matrix membranes (MMMs) based on polyurethane (PU) and silica nanoparticles was fabricated. Three types of PU were synthesized with different polyethylene glycol (PEG)/polytetramethylene glycol ratios (PU0, PU75, and PU100, the corresponding numbers: PEG percent). The MMMs were characterized using FTIR, SEM, and gas permeation measurements (N 2 , O 2 , CH 4 , and CO 2 ). The phase mixing of PU0 and PU75 membranes increased with silica content. This resulted in a decrease in gas permeability, while an increase in CO 2 /CH 4 and CO 2 /N 2 selectivities. The gas separation trend for PU100 membranes was the opposite. The molecular probing approach was served to study the interfacial structure of the MMMs. The existence of a rigidified polymer layer with 2.38 and 2.44 nm thicknesses around silica was revealed through PU0 and PU75 membranes, respectively. In contrary, silica was surrounded by a polymer chain dilution interphase with a thickness of 2.20 nm for PU100 MMMs.