Cold stress causes physiological dysfunction, tissue damage, and finally death in fishes, and increasing studies have suggested that epigenetic mechanisms play essential roles in the cold stress response in fishes. Our previous study showed that cold stress induced the production of reactive oxygen species (ROS) in zebrafish -derived ZF4 cells in a temperature and time-dependent manner and that the genomic DNA methylation level was increased under short-term (18℃ for 5 days) cold exposure and decreased under long-term cold exposure (18℃ for 30 days). However, the relationship among DNA methylation, ROS production, and cold acclimation in fishes remains poorly understood. In the present study, zebrafish ZF4 cells were exposed to short-term (18℃ or 10℃, for 3 or 5 days) and long-term (18℃ for 30 days) cold stress, and then the global DNA methylation level was detected by digestion with the methylation-sensitive enzyme I. The results showed that short-term cold stress caused remarkable growth arrest and cell death in ZF4 cells, and cold acclimation was observed under long-term cold stress. Additionally, global DNA methylation increased remarkably under short-term cold stress ( < 0.05). Co-treatment of ZF4 cells with -acetylcysteine inhibited global DNA methylation induced by short-term cold stress ( < 0.05), suggesting that ROS affects short-term cold stress-induced global DNA methylation levels. Co-treatment of ZF4 cells with the ataxia telangiectasia mutated (ATM) inhibitor KU-55933 also inhibited the induction of global DNA methylation under short-term cold exposure, indicating the involvement of DNA damage repair pathways in this process. Our data indicate that short-term cold stress resulted in ROS production and ataxia telangiectasia mutated activation, which then up-regulated global DNA methylation in ZF4 cells. The present study improves our understanding of the role of DNA methylation under cold stress in fishes.