Summary Iron deficiency anemia (IDA) is one of the most serious forms of malnutrition. This experiment was conducted to investigate whether acidic xylooligosaccharide (U-XOS), expected to have a high iron bioavailability, was useful in the prevention of iron deficiency. Experiment 1: Nineteen female Sprague-Dawley rats (20 wk old) were fed three different diets for 28 d; a U-XOS-supplemented low-iron diet (LI-X, n ϭ 7), a low-iron diet (LI, n ϭ 6), and a control diet (C, n ϭ 6). On day 28, the LI-X and LI groups showed iron deficiency without anemia. A significant difference in the total and unsaturated iron binding capacity, and serum transferrin saturation level was shown in the LI-X and LI groups, compared with the C group. However, the decrease of hepatic iron content of the LI-X group was suppressed compared with the LI group. Experiment 2: Eleven male Sprague-Dawley rats (7 wk old) were fed a U-XOS-supplemented diet (X, n ϭ 5) or a control diet (C, n ϭ 6) for 7 d. No significant difference in body weight gain or food intake was demonstrated between the two groups; the apparent iron absorption rate of the X group increased clearly compared with that of the C group. These results suggested that a U-XOS diet could preserve storage of hepatic iron in adult female rats fed a low-iron diet and could prevent IDA by promotion of dietary iron absorption, inhibition of iron excretion, and/or improvement of iron bioavailability.