To evaluate the hypothesis that mild iron deficiency increases dependence upon gluconeogenesis, control and mildly iron-deficient (Hb = 80 + 2 g/L) rats were injected with mercaptopicolinic acid (MPA), a known inhibitor of gluconeogenesis, or with injection vehicle (sham) and studied at rest or after 30 min of treadmill running (13.4 m/min, 0% grade). Liver glycogen concentration was lower in resting iron-deficient rats than in resting control rats, but iron deficiency did not influence arterial substrates or hormones in sham-treated rats. Glucose and insulin concentrations were less in resting control and iron-deficient MPA-treated rats than in sham-treated animals. However, arterial lactate was greater in resting iron-deficient MPA-treated rats than control MPA-treated animals, and glucagon and epinephrine were greater in resting iron-deficient MPA-treated rats than in iron-deficient sham-treated animals, indicating that gluconeogenesis is more important to maintenance of euglycemia in resting iron-deficient animals than in controls. Moderate exercise stimulated glucose metabolism in iron-deficient rats, as evidenced by the lower arterial glucose and higher arterial lactate when compared with resting iron-deficient rats. However, MPA treatment did not clearly establish differences between iron-deficient and control rats after exercise. Therefore, changes in substrate and hormone concentrations in resting iron-deficient MPA-treated rats indicate that dependence on gluconeogenesis for maintenance of euglycemia is greater at rest with dietary iron deficiency. Furthermore, consistent with previously published results for severely iron-deficient rats, results from the present investigation indicate that dependence on glucose metabolism is greater during moderate exercise in mildly iron-deficient rats.