This study reports the effects of a high-fat (HF) diet on the iron (Fe) status of growing rats over 8 weeks. Tissue Fe levels were analysed by atomic absorption spectrophotometry, and whole-body adiposity was measured by dual-energy X-ray absorptiometry. Histopathology and morphometry of adipose tissue were performed. Liver homogenates were used for measuring ferroportin (Fpn)-1 protein levels by immunoblotting, and transcript levels were used for Fe genes measured by real-time PCR. Tissue Fe pools were fit to a compartmental biokinetic model in which Fe was assessed using 14 compartments and 27 transfer constants (kj,i from tissue “i” to tissue “j”) adapted from the International Commission on Radiological Protection (ICRP) 69. Ten kj,i were calculated from the experimental data using nonlinear regression, and 17 were estimated by allometry according to the formula kj,i = a · Mb. Validation of the model was carried out by comparing predicted and analysed Fe pool sizes in red blood cells (RBCs), the liver and the spleen. Body adiposity was negatively associated with serum Fe levels and positively associated with liver Fe stores. An inferred increase in Fe transfer from bone marrow to the liver paralleled higher hepatic Fe concentrations and ferritin heavy-chain mRNA levels in the HF diet-fed animals, suggesting that liver Fe accumulation occurred at least in part due to a favoured liver RBC uptake. If this feeding condition were to be prolonged, impaired Fe decompartmentalization may occur, ultimately resulting in dysmetabolic Fe overload.