We hypothesized that iron content and regulatory factors, which may be involved in exercise tolerance, are differentially expressed in systemic and muscle compartments in iron deficient severe chronic obstructive pulmonary disease (COPD) patients. In the vastus lateralis and blood of severe COPD patients with/without iron depletion, iron content and regulators, exercise capacity, and muscle function were evaluated in 40 severe COPD patients: non-iron deficiency (NID) and iron deficiency (ID) (20 patients/group). In ID compared to NID patients, exercise capacity, muscle iron and ferritin content, serum transferrin saturation, hepcidin-25, and hemojuvelin decreased, while serum transferrin and soluble transferrin receptor and muscle IRP-1 and IRP-2 increased. Among all COPD, a significant positive correlation was detected between FEV1 and serum transferrin saturation. In ID patients, significant positive correlations were detected between serum ferritin, hepcidin, and muscle iron content and exercise tolerance and between muscle IRP-2 and serum ferritin and hepcidin levels. In ID severe COPD patients, iron content and its regulators are differentially expressed. A potential crosstalk between systemic and muscle compartments was observed in the ID patients. Lung function and exercise capacity were associated with several markers of iron metabolism regulation. Iron status should be included in the overall assessment of COPD patients given its implications in their exercise performance.