We developed highly selective polyurethane (PU) membranes with incorporated NiO nanoparticles for gas-phase carbon dioxide separation. PU was synthesized with polytetramethylene glycol and isophoronediisocyanate, and 1,4-butandiamine/1,4-butandiol as the chain extender (1:3:2 molar ratio). Mixed matrix membranes (MMMs) composed of PU and NiO nanoparticles were fabricated using the solution casting method. Scanning electron microscopy (SEM) confirmed the even distribution of NiO in the PU matrix at the nanoscale. Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) were used to examine the phase separation between the soft and hard domains of PU, and the distribution of NiO nanoparticles in both domains. DSC and X-ray diffraction (XRD) pattern confirmed that the crystalline structures in both soft and hard segments are changed by NiO incorporation. The effects of NiO nanoparticles on the gas permeability, diffusivity, and solubility of pure CO2, CH4, O-2, and N-2 gases were studied at various temperatures and feed pressures. A large increase in the CO2/N-2 permselectivity with a simultaneous reduction in the gas permeability of CH4, O-2, and N-2 was observed. When the NiO content was low (1 wt%), the CO2/N-2 permselectivity increased by 79.21%, and the CO2 permeability increased by 1.57%. Increasing the nanoparticle load to 5 wt% increased the CO2/N-2 permselectivity by 161.1%, while the CO2 permeability was decreased by 3.31%.