β-thalassemia caused by the C→T mutation at nucleotide 654 of the intron 2 (β(IVSII-654)) results in aberrant splicing of β-globin RNA, leading to an almost absence of β-globin synthesis. Although trabecular and cortical bone loss was previously reported in β-thalassemic mice with deletion of β-globin gene, the microscopic changes in trabecular structure in β(IVSII-654) thalassemic mice remained elusive. Here, we investigated the macroscopic and microscopic bone changes in 12-week-old β(IVSII-654) knockin thalassemic mice by dual-energy X-ray absorptiometry (DXA) and histomorphometric analysis, respectively. DXA revealed a decrease in bone mineral density in the lumbar vertebrae and tibial metaphysis, but not in the femoral diaphysis, suggesting that β(IVSII-654) thalassemia predominantly led to osteopenia at the trabecular site, but not the cortical site. Further histomorphometric analysis of the tibial secondary spongiosa showed that trabecular bone volume was significantly decreased with the expansion of marrow cavity. Decreases in osteoblast surface, osteoid surface, mineral apposition rate, mineralizing surface, and mineralized volume were also observed. Moreover, trabecular bone resorption was markedly enhanced as indicated by increases in the osteoclast surface and eroded surface. It could be concluded that β(IVSII-654) thalassemia impaired bone formation and enhanced bone resorption, thereby leading to osteopenia especially at the trabecular sites, such as the tibial metaphysis.