The H-2 production potential of [FeFe]-hydrogenase, a hydrogen-producing enzyme from green algae, is reported to be promising for economical and large-scale production of H-2 as an alternative source of renewable energy. The production of hydrogen takes place at the catalytic center buried in the enzyme core. Unfortunately, binding of O-2 to the catalytic center of the enzyme irreversibly inactivates it, essentially blocking hydrogen production. Therefore, a better understanding of the mechanism of O-2 entry/exit is necessary to develop strategies for designing oxygen-tolerant enzymes. In this work, we investigated the pathways and diffusion channels of O-2 gas in this hydrogenase. Through exhaustive mapping of oxygen-diffusion channels, we computed a full thermodynamic map of preferred binding locations of O-2 gas within the enzyme interior, which showed that O-2 can enter and exit the enzyme through multiple pathways along which are key residues that are known to perturb rates of O-2 binding. The global minimum in the free-energy landscape is located near the H-cluster, a key metallic center within the enzyme. Along O-2 diffusion channels, we further identified several residues that could be potential candidates for mutations to increase the oxygen tolerance of [FeFe]-hydrogenase.