Background: Acute kidney injury (AKI) is associated with dysregulated iron metabolism, which may play a significant role in cellular injury. The effect of hemodialysis (HD) on iron metabolism in AKI therapy has not been well defined. Methods: The effects of HD on iron parameters were tested in control rats and bilateral nephrectomy (BNx) rats. The BNx rats were divided into the following 3 groups: 1) the Sham-operated group (BNx-Sham), 2) the BNx group, and 3) the HD group (BNx-HD), which received HD therapy 40-45 hours after BNx. Sections of the liver or spleen were stained with Berlin blue to examine the accumulation of iron. The mRNA levels of hepcidin and ferroportin1 in the spleen and liver were also quantified using RT-PCR. Results: In the BNx group, the plasma iron and hematocrit levels were decreased, and hepcidin levels were increased. The iron staining in the spleen in the BNx group was significantly more intense than that in the BNx-Sham group; however, after an HD session, splenic iron staining diminished to the level of the Sham group along with an increase in plasma iron and a decrease in hepcidin. Conclusions: BNx moved iron from hemoglobin and the plasma to the spleen, which is associated with an increase in plasma hepcidin. A single HD session accelerated the release of iron from the spleen, and the increased plasma iron was linked to the removal of hepcidin. Our data suggested that hepcidin might dynamically modulate the iron metabolism in BNx as well as in HD.