Iron-based enzymes efficiently activate molecular oxygen to perform the oxidation of methane to methanol (MTM), a reaction central to the contemporary chemical industry. Conversely, a very limited number of artificial catalysts have been devised to mimic this process. Herein, we employ the MIL-100(Fe) metal-organic framework (MOF), a material that exhibits isolated Fe sites, to accomplish the MTM conversion using O2 as the oxidant under mild conditions. We apply a diverse set of advanced operando X-ray techniques to unveil how MIL-100(Fe) can act as a catalyst for direct MTM conversion. Single-phase crystallinity and stability of the MOF under reaction conditions (200 or 100 °C, CH4 + O2) are confirmed by X-ray diffraction measurements. X-ray absorption, emission, and resonant inelastic scattering measurements show that thermal treatment above 200 °C generates Fe(II) sites that interact with O2 and CH4 to produce methanol. Experimental evidence-driven density functional theory (DFT) calculations illustrate that the MTM reaction involves the oxidation of the Fe(II) sites to Fe(III) via a high-spin Fe(IV)═O intermediate. Catalyst deactivation is proposed to be caused by the escape of CH3• radicals from the relatively large MOF pore cages, ultimately resulting in the formation of hydroxylated triiron units, as proven by valence-to-core X-ray emission spectroscopy. The O2-based MTM catalytic activity of MIL-100(Fe) in the investigated conditions is demonstrated for two consecutive reaction cycles, proving the MOF potential toward active site regeneration. These findings will desirably lay the groundwork for the design of improved MOF catalysts for the MTM conversion.