DNA topoisomerase IIIα (TOP3A) is a highly conserved type IA topoisomerase critical for genome maintenance. Its deletion causes embryonic lethality in many organisms, which has hampered attempts to understand its physiological role. Recently, human subjects with TOP3A mutations were identified who display a Bloom's syndrome (BS)-like phenotype and mitochondrial dysfunction, consistent with TOP3A's roles in the nucleus, alongside the Bloom's helicase, and in mitochondria. Here, we generate a Top3a mutant mouse model mimicking those patient mutations that truncate the C-terminal domain (CTD). In contrast to humans, homozygous Top3a mutant mice lose viability at around 7.5 days post coitum (dpc). Mutant embryos and embryonic stem cells progressively lose mitochondrial DNA. Biochemical analyses indicate that CTD loss impairs DNA binding and plasmid relaxation activity and that the isolated CTD binds both single- and double-stranded DNA substrates. Our findings highlight the CTD's important role in TOP3A function in both the nucleus and mitochondria.