Testosterone stimulates iron‐dependent erythropoiesis and suppresses hepcidin. To clarify the role of iron in mediating testosterone's effects on erythropoiesis, we induced iron deficiency in mice by feeding low iron diet. Iron‐replete and iron‐deficient mice were treated weekly with testosterone propionate or vehicle for 3 weeks. Testosterone treatment increased red cell count in iron‐replete mice, but, surprisingly, testosterone reduced red cell count in iron‐deficient mice. Splenic stress erythropoiesis was stimulated in iron‐deficient mice relative to iron‐replete mice, and further increased by testosterone treatment, as indicated by the increase in red pulp area, the number of nucleated erythroblasts, and expression levels of TfR1, GATA1, and other erythroid genes. Testosterone treatment of iron‐deficient mice increased the ratio of early‐to‐late erythroblasts in the spleen and bone marrow, and serum LDH level, consistent with ineffective erythropoiesis. In iron‐deficient mice, erythropoietin levels were higher but erythropoietin‐regulated genes were generally downregulated relative to iron‐replete mice, suggesting erythropoietin resistance. Conclusion: Testosterone treatment stimulates splenic stress erythropoiesis in iron‐replete as well as iron‐deficient mice. However, testosterone worsens anemia in iron‐deficient mice because of ineffective erythropoiesis possibly due to erythropoietin resistance associated with iron deficiency. Iron plays an important role in mediating testosterone's effects on erythropoiesis.