Heterozygous β-globin gene knockout thalassemia (BKO) mice derived from C57BL/6 wild-type (WT) mice have phenotypic of β-thalassemia (BT) and have been widely used for studying this disease except reproductive disorders. The present study determined whether male BKO mice recapitulate reproductive problems as BT men. Mice were randomly assigned into groups depending on the genotype (WT vs. BKO) and intervention (control vs. iron-loaded). Euthanized mice were collected blood, testes, epididymides, hypothalamus, and anterior pituitary for assessing hematological parameters, plasma iron and testosterone levels, testis iron levels, sperm characteristics, and histological alterations. Iron administration caused significant increases of plasma and testis iron levels (p <
0.001) but had no significant influence on the hematological profile of BKO mice, which indeed had fewer erythrocyte, hemoglobin, and hematocrit but had greater reticulocyte than WT (p <
0.001 to p = 0.017). Furthermore, irrespective of the genotype, iron administration decreased plasma testosterone levels (p = 0.03 to p >
0.05), total sperm count (p <
0.001), and percent normal sperm morphology (p ≤ 0.01). Based on Perls' Prussian blue staining, excess iron was ubiquitously present in the anterior pituitary and testicular interstitium of iron-loaded mice. This mineral, however, caused no significant changes in reproductive organs microstructure as visualized by hematoxylin and eosin staining. In conclusion, besides physiological dysfunction of many organ systems, iron-loaded male BKO mice exhibit reproductive problems and abnormal sperm characteristics similar to BT men. Therefore, this animal model seems invaluable for future biomedical research involved in various aspects of BT-related male reproductive disorders.