Abstract: [FeFe] hydrogenases catalyze reversible hydrogen evolution at rates as high as 10,000 turnovers per second. This exceptional catalytic ability is very attractive for the use of hydrogenases in renewable energy applications and biohydrogen production. Unfortunately, these enzymes degrade irreversibly upon exposure to minute amounts of oxygen, presenting major roadblocks for study and implementation in practical or industrial applications. The recent finding of an oxygen-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA) is a long-awaited breakthrough in the field of enzymatic hydrogen catalysis because it presents an unprecedented opportunity to implement this very efficient enzyme into sustainable systems. We employed a multidisciplinary approach involving advanced EPR, FTIR and NRVS spectroscopy, electrochemistry, bioinformatics, molecular dynamics simulations, and density functional theory to investigate this extraordinary enzymatic system in detail. The presented work provides crucial details necessary to understand the mechanism of oxygen-tolerance and to determine the structural basis for this mechanism. Our sequence similarity analysis also suggests that this enzyme is a representative of a large group of [FeFe] hydrogenases, setting an exciting avenue for future studies of these enzymes.