Iron is an essential mineral for the body, and iron deficiency generally leads to anemia. However, because non-anemic iron deficiency can exist, we performed a comprehensive transcriptome analysis of the liver to define the effects of this condition on the body. Four-week-old male rats were fed a low-iron diet (approximately 3 ppm iron) for 3 days and compared with those fed a normal diet (48 ppm iron) by pair feeding as a control. The rats in the iron-deficient diet group developed a non-anemic iron-deficient state. DNA microarray analysis revealed that during this short time, this state conferred a variety of effects on nutrient metabolism in the liver. In comparison with long-term (17 days) iron-deficiency data from a previous study, some of the changed genes were found to be common to both short- and long-term iron deficiency models, some were specific to the short-term iron deficiency model, and the others were oppositely regulated between the two feeding terms. Taken together, these data suggest that although the blood hemoglobin level itself remains unchanged during non-anemic iron deficiency, a variety of metabolic processes involved in the maintenance of the energy balance are altered.