Increasing evidence suggests that the mitochondrial outer membrane protein mitoNEET is a key regulator of energy metabolism, iron homeostasis, and production of reactive oxygen species in mitochondria. Previously, we reported that mitoNEET is a redox enzyme that catalyzes electron transfer from the reduced flavin mononucleotide (FMNH2) to oxygen or ubiquinone via its unique [2Fe-2S] clusters. Here, we explore the reduction and oxidation kinetics of the mitoNEET [2Fe-2S] clusters under anaerobic and aerobic conditions. We find that the mitoNEET [2Fe-2S] clusters are rapidly reduced by a catalytic amount of FMNH2 which is reduced by flavin reductase and an equivalent amount of NADH under anaerobic conditions. When the reduced mitoNEET [2Fe-2S] clusters are exposed to air, the [2Fe-2S] clusters are slowly oxidized by oxygen at a rate constant of about 6.0M−1 s−1. Compared with oxygen, ubiquinone-2 has a much higher activity to oxidize the reduced mitoNEET [2Fe-2S] clusters at a rate constant of about 3.0×103 M−1 s−1 under anaerobic conditions. Under aerobic conditions, the mitoNEET [2Fe-2S] clusters can still be reduced by FMNH2 in the presence of flavin reductase and excess NADH. However, when NADH is completely consumed, the reduced mitoNEET [2Fe-2S] clusters are gradually oxidized by oxygen. Addition of ubiquinone-2 also rapidly oxidizes the pre-reduced mitoNEET [2Fe-2S] clusters and effectively prevents the FMNH2-mediated reduction of the mitoNEET [2Fe-2S] clusters under aerobic conditions. The results suggest that ubiquinone may act as an intrinsic oxidant of the reduced mitoNEET [2Fe-2S] clusters in mitochondria under aerobic and anaerobic conditions.