Objectives: Iron is involved in processes involving oxygen transfer and utilization. Excess iron is linked to cardiovascular diseases and some types of cancer. Iron overload is associated with oxidative stress development, and may have important interactions with lipid metabolism in the liver favoring the development and progression of non-alcoholic fatty liver disease. The aim of the study described here was to assess the effect of high intake of iron on oxidative stress-related parameters, lipid metabolism, and levels of long-chain polyunsaturated fatty acids (LCPUFAs) in liver and other tissues of the rat. Methods: Male Wistar rats (21 d old) were fed an iron-rich diet (200 mg iron/kg diet, IRD) versus a control diet (50 mg iron/kg diet; CD) for 21 d. Samples of erythrocytes, liver, adipose tissue, brain, heart, and testicles were evaluated for fatty acid composition and hepatic biochemical and oxidative stress parameters, D-6 and D-5 desaturase activities, SREBP-1c and PPAR-a mRNA expression and DNA-binding capacity, and lipolytic, lipogenic, and antioxidant enzymatic activities. Results: The IRD caused liver steatosis and increased activity of plasma transaminases, with higher oxidative stress status in plasma and liver. Liver D-6 and D-5 desaturase exhibited decreased activity, but enhanced expression in response to the IRD compared with the CD, with lower levels of u-3 and u-6 LCPUFAs and higher expression and DNA binding of SREBP-1c, whereas expression and DNA-binding activity of PPAR-a were diminished. Conclusions: IRD induced oxidative stress and a reduction in the desaturation capacity of the liver, with LCPUFA depletion in the different tissues studied, thus promoting a pro-steatotic condition in the liver.