This study aims to explore the damage mechanism of hemoglobin on Ctenopharyngodon idella kidney (CIK) cells. Firstly, the growth of CIK cells stimulated by hemoglobin and heme was observed, and the results showed that hemoglobin and heme significantly inhibited the growth of CIK cells. Secondly, the expression of iron metabolism-related genes in CIK cells was detected after stimulated by hemoglobin and hem. The results showed that the stimulation of hemoglobin and heme up-regulated the expression of iron metabolism-related genes to different degrees. The expression of inflammation and antioxidant-related enzymes in CIK cells after stimulated by hemoglobin and heme was also examined. The results suggested that hemoglobin and heme could activate the expression of inflammatory factors through NF-κB pathway, such as pro-inflammatory cytokines TNF-α, IL-1β and IL-6, the anti-inflammatory factor IL-10 and chemokines IL-4 and IL-8, and also activate the expression levels of three antioxidant enzymes, including Superoxide dismutase (SOD), Catalase (CAT) and Glutathione peroxidase (GSH-Px). In order to further detect the toxic effect of hemoglobin on CIK cells, this study also detected the intracellular iron and ROS in CIK cells after hemoglobin stimulation. The results showed that hemoglobin stimulation significantly increased intracellular iron and ROS. Finally, we also examined the expression of apoptosis-related genes in CIK cells after stimulated by hemoglobin and heme, and the results showed that both hemoglobin and heme significantly activated the expression levels of apoptosis-related genes in CIK cells. In conclusion, the results of this study suggest that the high oxidative activity of hemoglobin can activate the expression of iron metabolism- and inflammation-related genes, as well as increasing intracellular iron and ROS levels, leading to up-regulated expression of apoptosis genes in CIK cells.