Osteoporosis is one of the most frequent complications in patients with β-thalassaemia, a genetic disorder which generally results in abnormal haemoglobin formation and iron-overload syndrome, yet little is known about the mechanism. Recent studies indicate that iron-overload is related with impaired osteogenesis, which also affects the activation of macrophages ̶ the key player in innate immune response. Accumulating evidence indicates that macrophages play an indispensable role in osteogenesis by interacting with bone marrow stromal cells (BMSCs). This study aimed to investigate the impact of iron-overload on the interaction between macrophages and BMSCs and how this eventually may lead to thalassaemia-related osteoporosis. Our in vivo results showed that the bone morphology and microarchitecture were abnormal in β^IVSII-654 knock-in mice (animal model of β-thalassaemia), which was accompanied by decreased mRNA and protein levels of osteogenic markers in the bone marrow tissue. Iron deposition was found in the trabecular bone, which was associated with abnormally activated macrophages. The in vitro results showed that iron-overload significantly influenced the activation and phenotype of macrophages by affecting the autophagy/apoptosis processes. The secreted factors derived from iron-overload stimulated macrophages resulted in dysregulation of osteogenic markers in BMSCs and eventually led to impaired osteogenesis. In conclusion, our study reveals that iron-overload results in dysregulated macrophage activation, leading to impaired bone formation, which could be the mechanism of thalassaemia-related osteoporosis.