The high-temperature ammonia-injected technology is acknowledged as a promising denitrification method with great NO_x reduction potential. In this work, char reduction experiments and density functional theory (DFT) calculations were conducted to investigate the heterogeneous reduction mechanism of NO by ammonia (NH₃) synergism with char, focusing on the role of O₂. Experimental results indicate that O₂ has an inhibition effect on the NO reduction efficiency, whose detailed mechanisms can be inferred from a molecular perspective. Theoretical results reveal that both Langmuir-Hinshelwood (L-H) and Eley-Rideal (E-R) mechanisms exist in the NH₃-injected denitrification reactions, presenting significant differences in N₂, N₂O, and H₂O evolution. By comparison, the E-R mechanism governs the NH₃–NO reactions, which reduces barriers by up to 14.5 %. The presence of O₂ can lead to a significant increase in the energy barriers with a concomitant decrease in reaction rates within a certain temperature range, which however does not change the dominant mechanism governing the NH₃ and NO interactions. Although some controversies are found in the kinetic analysis when the temperature increases, the results here shed light on the NO heterogeneous reduction mechanisms with NH₃ at high temperature, which also guides the coal/NH₃ co-firing investigations.