Dioxygen (O ₂ ) and other gas molecules have a fundamental role in a variety of enzymatic reactions. However, it is only poorly understood which O ₂ uptake mechanism enzymes employ to promote efficient catalysis and how general this is. We investigated O ₂ diffusion pathways into monooxygenase and oxidase flavoenzymes, using an integrated computational and experimental approach. Enhanced-statistics molecular dynamics simulations reveal spontaneous protein-guided O ₂ diffusion from the bulk solvent to preorganized protein cavities. The predicted protein-guided diffusion paths and the importance of key cavity residues for oxygen diffusion were verified by combining site-directed mutagenesis, rapid kinetics experiments, and high-resolution X-ray structures. This study indicates that monooxygenase and oxidase flavoenzymes employ multiple funnel-shaped diffusion pathways to absorb O ₂ from the solvent and direct it to the reacting C4a atom of the flavin cofactor. The difference in O ₂ reactivity among dehydrogenases, monooxygenases, and oxidases ultimately resides in the fine modulation of the local environment embedding the reactive locus of the flavin.