Endoreduplication is a variant of the cell cycle in which DNA replication occurs without mitosis. The physiological role of endoreduplication in plants is, however, not yet fully elucidated. We have conducted flow cytometric analysis to investigate the effect of DNA double-strand breaks (DSBs) on endoreduplication in radish (Raphanus sativus var. longipinnatus) cotyledons, where the proportion of higher ploidy cells was increased by the treatment of DSBs-inducing agent Zeocin. To understand the microscopic details of the endoreduplication caused by the Zeocin-induced DSBs, in this study, we examined nuclear DNA content and cell size in epidermal cells of radish cotyledons after Zeocin treatment. Enlargement of the cell size and puzzled-shape of the epidermal pavement cells was compromised by Zeocin treatments. Fluorocytometric analysis showed that the nuclear DNA distribution of the Zeocin-treated epidermis was distinct from that of the untreated epidermis; that is, a frequency of the nuclei with higher ploidy in the Zeocin-treated epidermis was higher than that in the untreated epidermis. A positive correlation between cell size and nuclear DNA content was found in both Zeocin-treated and -untreated epidermis, but that in the Zeocin-treated epidermis was less relevant than that in the untreated cotyledons. These results suggest that the Zeocin-stimulated ectopic endoreduplication compromises cell expansion during epidermal cell development.