A marked decrease in β-globin production led to β-thalassemia, a hereditary anemic disease associated with bone marrow expansion, bone erosion, and osteoporosis. Herein, we aimed to investigate changes in bone mineral density (BMD) and trabecular microstructure in hemizygous β-globin knockout thalassemic (BKO) mice, and to determine whether endurance running (60 min/day, 5 days/week for 12 weeks in running wheels) could effectively alleviate bone loss in BKO mice. Both male and female BKO mice (1-2 months old) showed growth retardation as indicated by smaller body weight and femoral length compared to their wild-type littermates. A decrease in BMD was more severe in female than male BKO mice. Bone histomorphometry revealed that BKO mice had decreases in trabecular bone volume, trabecular number, and trabecular thickness, presumably due to suppression of osteoblast-mediated bone formation and activation of osteoclast-mediated bone resorption, the latter of which was consistent with elevated serum levels of osteoclastogenic cytokines, interleukin-1α and -1β. As determined by peripheral quantitative computed tomography, running increased cortical density and thickness in the femoral and tibial diaphyses of BKO mice when compared to those of sedentary BKO mice. Several histomorphometric parameters suggested an enhancement of bone formation (e.g., increased mineral apposition rate) and suppression of bone resorption (e.g., decreased osteoclast surface), which led to increases in trabecular bone volume and trabecular thickness in running BKO mice. In conclusion, BKO mice exhibited pervasive osteopenia and impaired bone microstructure, while running exercise appeared to be an effective intervention in alleviating bone microstructural defect in β-thalassemia.