ObjectiveMiddle-aged women with obesity are at increased risk of iron overload and iron disorder is known to disrupt n-3 polyunsaturated fatty acid homeostasis. We evaluated relationships between pretreatment hemoglobin and n-3 polyunsaturated fatty acid levels, and tested whether pretreatment hemoglobin contributed to inter-individual variability in weight loss with special focus on changes in body weight, iron and n-3 polyunsaturated fatty acid profiles. Study design117 middle and older aged women with obesity and more than two metabolic abnormalities were randomized to a 12-week hypocaloric diet without or with fish oil supplementation. Blood iron biomarker and erythrocyte membrane phospholipid profiles were evaluated. Main outcomeThe absolute change from baseline to week 12 in serum iron and erythrocyte n-3 polyunsaturated fatty acid levels according to pretreatment hemoglobin tertiles and fish oil supplementation. ResultsA Pearson correlation analysis showed that pretreatment hemoglobin levels were negatively correlated with linoleic acid (r = −0.231), α-linoleic acid (r = −0.279), and n-3 polyunsaturated fatty acid (r = −0.217) (all p < 0.05). Dietary weight loss markedly enhanced erythrocyte membrane lipids of linoleic acid, α-linoleic acid, and n-6 and n-3 polyunsaturated fatty acid only in those women with the highest pretreatment hemoglobin levels (tertile 3) (all p < 0.05). Fish oil supplementation increased bioavailable iron in women with moderate pretreatment hemoglobin levels (tertile 2) (p < 0.05) and, to a lesser extent, prevented a reduction in circulating iron in those with the lowest hemoglobin levels (tertile 1). ConclusionDietary weight loss is an effective treatment program to manage obesity-related iron and n-3 polyunsaturated fatty acid disorders, particularly for middle-aged women with obesity and iron overload.