In our previous study, we compared the transcriptome between Antarctic fish and temperate fish and found that genes were involved in reactive oxygen species (ROS) scavenge and Mitogen-activated protein kinase (MAPK) pathway associated with the cold adaptation in fish. To further study the regulatory network and the reactive oxygen species (ROS) homeostasis under cold stress in fish, we examined the level of ROS and the expression of stress-related proteins in zebrafish-derived ZF4 cells exposed to mildly and severely cold temperatures, 18℃ and 10℃, at various time points (1 d, 3 d, and 5 d). Results showed that (1) DCFH-DA probe method was used to determine the level of ROS in cells. We found that the level of ROS in cell positively correlated with the intensity of cold stress. After 3 days of cold treatment, the level of ROS in cells significantly increased to 1.23±0.04 (0.05) times, respectively, at 18℃ and 10℃, compared with that in cells at 28℃. (2) Western blot showed that the expression of MAPKs (p-JNK p54, p-JNK p46 and p-p38) was induced under cold stress, and reached the highest at 3 d of 10℃. (3) In addition, the expression of γH2A.X reached peak after 3 days both at 18℃ and 10℃. These findings showed that cold temperature could induce the production of ROS in ZF4 cells. The level of ROS in ZF4 cells is dependent on the stress intensity and duration. We observed the markedly induction of ROS in the cells at the first 3 days of cold treatment, while the expression of p-JNK, p-p38, and γH2A.X was markedly induced at the 3 d, indicating that 3 d of cold treatment might be a key time point to determine the protein expression in ZF4 cells.