Significance The liver is rich in mitochondria and has an exceptional regenerative capacity after partial hepatectomy or transplantation, viral infections, or chemical injuries; however, relatively little is known about the genetic factors for mitochondrial DNA (mtDNA) replication during liver regeneration. Here, we show that liver regeneration is markedly reduced in mice lacking mitochondrial topoisomerase I ( TOP1mt ). This defect is linked with reduced production of mtDNA and defective mitochondrial functions during acute energy demand for liver regeneration. Additionally, TOP1mt KO primary hepatocytes from CCl ₄ -treated mice showed reduced and damaged mitochondria, decreased O ₂ consumption, and ATP production. Together with mtDNA depletion and regeneration experiments with ethidium bromide, these results demonstrate that Top1mt is required for mtDNA synthesis and appropriate liver regeneration.