Diet is the primary source of iron (Fe) for freshwater fish, and the absorption of Fe is believed to occur via the Nramp family of divalent metal transporters (also called DMT1). Presently, the homeostatic regulation of dietary Fe absorption in fish is poorly understood. This study examined the gastrointestinal mRNA expression of two Nramp isoforms, Nramp-β and Nramp-γ, in the freshwater rainbow trout (Oncorhynchus mykiss), following exposure to elevated dietary Fe [1,256 mg Fe/kg food vs. 136 mg Fe/kg food (control)] for 14 days. The physiological performance, plasma Fe status and tissue-specific accumulation of Fe were also evaluated. In general, the mRNA expression level of Nramp was higher in the intestine relative to the stomach. Interestingly, fish fed on a high-Fe diet exhibited a significant induction in Nramp expression after 7 days, followed by a decrease to the level observed in control fish on day 14. The increase in Nramp expression correlated with the elevated gastrointestinal and plasma Fe concentrations. However, the hepatic Fe concentration remained unchanged during the entire exposure period, indicating strong homeostatic regulation of hepatic Fe level in fish. Fish appeared to handle increased systemic Fe level by elevating the plasma transferrin level, thereby enhancing the Fe-binding capacity in the plasma. Overall, our study provides new interesting insights into the homeostatic regulation of dietary Fe uptake and handling in freshwater fish.