Topoisomerases are critical for replication, DNA packing and repair, as well as for transcription by allowing changes in DNA topology. Cellular DNA is present both in nuclei and mitochondria, and mitochondrial topoisomerase I (Top1mt) is the only DNA topoisomerase specific for mitochondria in vertebrates. Here, we report in detail the generation of TOP1mt knockout mice, and demonstrate that mitochondrial DNA (mtDNA) displays increased negative supercoiling in TOP1mt knockout cells and murine tissues. This finding suggested imbalanced topoisomerase activity in the absence of Top1mt and the activity of other topoisomerases in mitochondria. Accordingly, we found that both Top2α and Top2β are present and active in mouse and human mitochondria. The presence of Top2α-DNA complexes in the mtDNA D-loop region, at the sites where both ends of 7S DNA are positioned, suggests a structural role for Top2 in addition to its classical topoisomerase activities.