The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix-localized Kaede protein (mt-Kaede) to analyze the dynamics of mitochondrial fission in BY-2 suspension cells. Analysis of the photoactivatable fluorescence of mt-Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY-2 suspension cells at various stages. Cellular proteins interacting with Myc-tagged dynamin-related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti-Myc antibody-conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time-of-flight mass spectrometry (CapLC Q-TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time-lapse imaging of the fluorescence of Dendra2-tagged AtDRP3A/3B after green-to-red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell-cycle transitions in BY-2 suspension cells.