To study the effects of acute stress reaction on iron concentration and iron homeostasis in Megalobrama amblycephala, we stimulated stress response by intraperitoneal injection of cortisol. The iron content in serum and liver was determined by spectrophotometer, and the iron homeostasis related gene (hepcidin & transferrin) expression were detected by quantitative real-time PCR at 0, 2, 4, 8, 10, 12, 24 and 48 h after cortisol injection. The results showed that the content of serum cortisol immediately increased significantly after injection, almost 10 folds compared with the control group, and gradually decreased backward. The content of serum iron in experimental group is higher than that in control group at 4, 8, 10 and 12 hours post injection (hpi), but the content of liver iron led to the opposite results. Hepcidin mRNA expression in liver rose remarkably, reaching the maximum at 8 hpi and 10 hpi, respectively, and then descended to the normal level. The transferrin showed similar expression pattern, and reached the peak at 10 hpi. The result indicated that large release of intracellular iron into body fluid led to increased risk of pathogenic infection and proliferation in vivo. Hepcidin and Tf were involved in regulation of iron metabolism and inflammatory response under stress in M. amblycephala and may play important roles in iron homeostasis and innate immune system.