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It is a big challenge to separate N2O from CO2 using adsorption because they have similar physical properties. The Fe3+–F− site in MIL-100Fe transforms to an unsaturated Fe2+ site under high-temperature activation (300 °C), and the target sorbent MIL-100Fe-300 exhibits the biggest difference in CO2 and N2O adsorption capacity, and the selectivity of N2O/CO2 (50%/50%) is up to 3.00 at 298 K. According to DFT calculations, the original Fe3+–F− site has strong interaction with CO2, but the open Fe2+ site has a stronger interaction with N2O. Through a breakthrough experiment, it was confirmed that MIL-100Fe-300 has the best N2O/CO2 separation performance, making it potentially a useful material in industry. [ABSTRACT FROM AUTHOR]