Fe-exchanged zeolites are heterogeneous catalysts that can potentially ensure simultaneous conversion of nitrous oxide (N2O) and nitric oxide (NO) using ammonia (NH3) as a selective reducing agent through their selective catalytic reduction reaction (N2O-NO-SCR). In this study, we rationalize the origin of the beneficial effect of N2O on the NO conversion by combining catalytic experiments with ex situ characterization and in situ/operando X-ray absorption spectroscopy (XAS) and infrared spectroscopy in diffuse reflectance mode (DRIFTS) on a series of Fe-ZSM-5 catalysts where we attempted to control Fe speciation at constant Fe content. The catalytic activity data revealed that N2O can promote NO conversion at different temperatures and to different extents. This behavior was found to be related to the activity of the catalysts in the NO-mediated N2O decomposition reaction, which ensures the oxidative transformation of NO and thus sustains the N2O-NO-SCR chemistry. The oxidation activity is in turn determined by processes of N2O activation and NO adsorption, which are a function of the Fe speciation and are likely catalyzed by a minority of isolated Fe2+ sites coordinated in different cationic environments. In agreement, the concentrations of the Fe species able to activate N2O (C alpha) and of the Fe species able to coordinate NO (C Fe-NO) decrease with an increasing degree of Fe agglomeration and govern especially the promotion of the NO conversion induced by N2O in this dual-site mechanism. Maximization of the concentration of both species is therefore essential to design Fe-exchanged zeolites with the highest activity toward the N2O-NO-SCR reaction.