We have studied a naturally occurring small-molecule antimitotic called diazonamide A. Diazonamide A is highly effective at blocking spindle assembly in mammalian cell culture and does so through a unique mechanism. A biotinylated form of diazonamide A affinity purifies ornithine delta-amino transferase (OAT), a mitochondrial enzyme, from HeLa cell and Xenopus egg extracts. In the latter system, the interaction between diazonamide A and OAT is regulated by RanGTP. We find that specific OAT knockdown in human cervical carcinoma and osteosarcoma cells by RNA interference blocks cell division and causes cell death, the effects largely phenocopying diazonamide A treatment in these cell lines. Our experiments reveal an unanticipated, paradoxical role for OAT in mitotic cell division and identify the protein as a target for chemotherapeutic drug development.