[FeFe] hydrogenases are enzymes capable of producing and oxidizing H-2 at staggering submillisecond time scales. A major limitation in applying these enzymes for industrial hydrogen production is their irreversible inactivation by oxygen. Recently, an [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) was reported to regain its catalytic activity after exposure to oxygen. In this report, we have determined that artificially matured CbHydA1 is indeed oxygen tolerant in the absence of reducing agents and sulfides by means of reaching an O-2-protected state (H-inact). We were also able to generate the H-inact state anaerobically via both chemical and electrochemical oxidation. We use a combination of spectroscopy, electrochemistry, and density functional theory (DFT) to uncover intrinsic properties of the active center of CbHydA1, leading to its unprecedented oxygen tolerance. We have observed that reversible, low-potential oxidation of the active center leads to the protection against O-2-induced degradation. The transition between the active oxidized state (H-ox) and the H-inact state appears to proceed without any detectable intermediates. We found that the H-inact state is stable for more than 40 h in air, highlighting the remarkable resilience of CbHydA1 to oxygen. Using a combination of DFT and FTIR, we also provide a hypothesis for the chemical identity of the H-inact state. These results demonstrate that CbHydA1 has remarkable stability in the presence of oxygen, which will drive future efforts to engineer more robust catalysts for biofuel production.