The TOP3A gene encodes protein products that are targeted to both the nucleus and to mitochondria. The nuclear isoform of TOP3A is required for the resolution of double Holliday junctions during homologous recombination, while the mitochondrial isoform of TOP3A separates hemicatenated daughter mitochondrial DNA molecules following DNA replication. We have sought to identify differences between the two protein isoforms, and find that the mitochondrial isoform of TOP3A is truncated. This truncation is the result of proteolytic cleavage within the mitochondrial matrix that removes approximately 90 amino acids from the protein C-terminus. We identify MPP as the protease responsible and reconstitute the cleavage reaction in vitro, finding that the reaction can be blocked by mutation of the cleavage site. Removal of the C-terminus of TOP3A alters the biochemical activity of the enzyme, resulting in greater ssDNA binding and decatenation activity relative to the full-length protein. We propose that the truncation of TOP3A within mitochondria provides an adaptation of the enzyme that favours its ability to decatenate mtDNA substrates within mitochondria.