Nitrous oxide (N2O) is a potent greenhouse gas that accumulates in the atmosphere due to anthropogenic N2O emissions, disrupting the nitrogen balance. N2O reductase (N2OR) in denitrifying bacteria contributes to the nitrogen cycle by converting N2O to molecular nitrogen as a last step. For the reduction of N2O during denitrification, electron donors must supply two electrons. This review discusses the in vivo physiological electron donors involved in the reduction reaction of N2OR: cytochrome c55X and pseudoazurin, as well as the non-physiological electron donors commonly used in N2OR studies: reduced MV/BV, dithionite, and ascorbate. The kinetic parameters of the connection between N2OR and the electron donors are also included. This aim of this review to gain further insight into the reduction mechanism of N2OR, presenting the electron transfer center, CuA, and the catalytic center, CuZ, of N2OR. The state changes of Cu site have a significant impact on electron transfer and N2O binding. Moreover, the review focuses on potential electron transfer pathways and binding sites in the electron donor → CuA → CuZ process, along with the steady-state turnover in the CuZ site. Additionally, the review explains the commonly used methods in mechanistic studies of N2OR. Modulating the electron transfer pathways of N2OR holds promise as an approach to decreasing N2O emissions.Graphical abstract: