β-thalassemia, a hereditary anemic disorder, is often associated with skeletal complications that can be found in both male and female. The present study aimed to investigate the age- and sex-dependent changes in bone mineral density (BMD) and trabecular microstructure in β^IVSII-654 knockin thalassemic mice. Dual energy X-ray absorptiometry (DXA) and computer-assisted bone histomorphometry was employed to investigate temporal changes in BMD and histomorphometric parameters in male and female β^IVSII-654 knockin mouse model of human β-thalassemia, in which impaired splicing of β-globin transcript was caused by hemizygous C->T mutation at nucleotide 654 of the intron 2. Young growing β^IVSII-654 mice (1 month old) manifested shorter bone length and lower BMD than their wild-type littermates, indicating possible growth retardation and osteopenia, the latter of which persisted until 8 months of age (adult mice). Interestingly, two-way analysis of variance suggested an interaction between gender and β^IVSII-654 genotype, i.e., more severe osteopenia in adult female mice. Bone histomorphometry further suggested that low trabecular bone volume in male β^IVSII-654 mice, particularly during a growing period (1-2 months), was primarily due to suppression of bone formation, whereas both low bone formation rate and a marked increase in osteoclast surface were observed in female β^IVSII-654 mice. In conclusion, osteopenia and trabecular microstructural defects were present in both male and female β^IVSII-654 knockin thalassemic mice, but the severity, disease progression and cellular mechanism differed between the two genders.