Membrane-based gas separation technology offers significant advantages compared to conventional processes. To enhance membrane performance, the incorporation of nanoparticles into the polymer matrix to fabricate mixed matrix membranes (MMMs) plays a pivotal role in the advancement of membrane-based gas separation technology. In this study, molybdenum oxide (MoO3) nanoparticles were incorporated into Pebax® 1657 MMMs for CO2 separation. The size distribution of nanoparticles was characterized using dynamic light scattering. Scanning electron microscope was used to ensure the even distribution of MoO3 nanoparticles within the polymer matrix, and all prepared membranes’ crystallinity was under scrutiny by X-ray diffraction and differential scanning calorimetry. Also, by employing Fourier-transform infrared spectroscopy, the Pebax® 1657-MoO3 MMMs were confirmed to be properly fabricated. It appears that MoO3 nanoparticles are attracted to both soft and rigid Pebax® segments by hydrogen bonds, contributing to favorable interfacial adhesion. Gas separation tests demonstrated that the Pebax® membrane incorporating 3 wt% MoO3 delivered the highest CO2 permeability of 209.5 barrer and the superior CO2/N2 selectivity of 245 at 10 bar and 35 ℃. Finally, CO2 permeability and CO2/N2 selectivity were improved by 73.43 and 217.9% compared to the pure Pebax® membrane.