This study investigates the role of dynamin-related GTPase Drp1 in mitochondrial fission during mitosis. Mitochondria in interphase HeLa cells form a long tubular network, but they fragment in early mitosis and then re-form in daughter cells. Drp1 knockdown cells maintain tubular mitochondrial structures during mitosis, indicating that Drp1 is essential for mitochondrial fragmentation in mitosis. Drp1 is specifically phosphorylated on Ser-585 by Cdk1/cyclin B during mitosis, and exogenous expression of an unphosphorylated mutant Drp1S585A reduces mitotic mitochondrial fragmentation. These findings suggest that phosphorylation of Drp1 on Ser-585 promotes mitochondrial fission in mitotic cells. The study provides evidence for the first time that Drp1-dependent mitochondrial fragmentation occurs in the early mitotic phase, contributing to the inheritance of mitochondrial morphology to daughter cells.This study investigates the role of dynamin-related GTPase Drp1 in mitochondrial fission during mitosis. Mitochondria in interphase HeLa cells form a long tubular network, but they fragment in early mitosis and then re-form in daughter cells. Drp1 knockdown cells maintain tubular mitochondrial structures during mitosis, indicating that Drp1 is essential for mitochondrial fragmentation in mitosis. Drp1 is specifically phosphorylated on Ser-585 by Cdk1/cyclin B during mitosis, and exogenous expression of an unphosphorylated mutant Drp1S585A reduces mitotic mitochondrial fragmentation. These findings suggest that phosphorylation of Drp1 on Ser-585 promotes mitochondrial fission in mitotic cells. The study provides evidence for the first time that Drp1-dependent mitochondrial fragmentation occurs in the early mitotic phase, contributing to the inheritance of mitochondrial morphology to daughter cells.