The N2O-mediated oxidative dehydrogenation of propane over iron-containing MFI zeolites was studied by in situ diffuse reflectance Fourier transform infrared spectroscopy, complemented by analyses in a tapered element oscillating microbalance coupled with gas chromatography and characterization by X-ray diffraction, transmission electron microscopy, and N2 adsorption. Samples with different iron speciation and acidic properties, induced by the preparation method (ion-exchange and steam activation) and the framework composition (Fe–Al–Si, Fe–Ga–Si, and Fe–Ge–Si) were analyzed. Real-time monitoring of the reaction under operando conditions allowed us to gain insight into the mechanism and kinetics of deactivation and coke formation, as well as zeolite regeneration in air. Deactivation of iron zeolites in the oxidative dehydrogenation of propane is caused by coke deposition on active extra-framework iron species. The mechanism of coke formation and the nature of the deposits were similar over the samples, independent of the catalytic performance and deactivation pattern. Regeneration in air led to the disappearance of coke-related bands and the practically complete recovery of the original porosity, while the zeolite crystallinity remained intact. The initial propylene yield over the fresh and regenerated Fe–Ga–Si zeolites was the same, but the latter sample exhibited faster deactivation. This is attributed to a change of iron constitution in the first reaction–regeneration cycle. The alteration of the local environment of the iron species was indirectly concluded from the appearance of a band associated with Lewis centers in the infrared spectra of the regenerated zeolite. In support of this, TEM studies demonstrated an extensive degree of iron clustering as FeO x nanoparticles in the regenerated catalyst compared with the fresh and coked catalysts.