Acute hypoxia in water has always been a thorny problem in aquaculture, and iron metabolism plays an important role in hypoxic stress in fish. To explore the effect of iron concentration on hypoxic stress in fish, Ammonium ferric citrate (FAC) and Deferoxamine (DFO) were used to supplement exogenous iron and remove endogenous and exogenous iron in ZFL cells, so as to compare the response of ZFL cells to hypoxic stress at different iron concentrations. The results showed that hypoxic stress significantly reduced the proliferation ability and survival rate of ZFL cells and the expression of iron metabolism-related genes, while supplementation of exogenous iron significantly enhanced the proliferation ability and survival rate of ZFL cells under hypoxic stress compared with the control group and the group of scavenging of endogenous and exogenous iron. Meanwhile, the mRNA expression levels of iron absorption (tfa), iron storage (fthl27, fthl28, fthl31) and iron output (fpn) were significantly increased, when ZFL cells under hypoxic stress were supplemented with exogenous iron. Furthermore, the detection of reactive oxygen species (ROS) levels in ZFL cells under hypoxic stress showed that supplemental exogenous iron could restore ROS levels in ZFL cells to a certain extent, indicating that the increase of intracellular ROS level in a certain range is beneficial to the resistance of ZFL cells to hypoxic stress. In conclusion, this study found that when ZFL cells were in acute hypoxia, supplemental exogenous iron ions could increase intracellular iron level, thus producing certain beneficial ROS and contributing to improving their survival rate in hypoxia environment.