The monoamine serotonin (5-HT), a well-known neurotransmitter, is also important in peripheral tissues. Several studies have suggested that 5-HT is involved in bone metabolism. Starting from our original observation of increased 5-HT_2B receptor (5-HT_2BR) expression during in vitro osteoblast differentiation, we investigated a putative bone phenotype in vivo in 5-HT_2BR knockout mice. Of interest, 5-HT_2BR mutant female mice displayed reduced bone density that was significant from age 4 months and had intensified by 12 and 18 months. This histomorphometrically confirmed osteopenia seems to be due to reduced bone formation because 1) the alkaline phosphatase-positive colony-forming unit capacity of bone marrow precursors was markedly reduced in the 5-HT_2BR mutant mice from 4 to 12 months of age, 2) ex vivo primary osteoblasts from mutant mice exhibited reduced proliferation and delayed differentiation, and 3) calcium incorporation was markedly reduced in osteoblasts after 5-HT_2BR depletion (produced genetically or by pharmacological inactivation). These findings support the hypothesis that the 5-HT_2BR receptor facilitates osteoblast recruitment and proliferation and that its absence leads to osteopenia that worsens with age. We show here, for the first time, that the 5-HT_2BR receptor is a physiological mediator of 5-HT in bone formation and, potentially, in the onset of osteoporosis in aging women.—Collet, C., Schiltz, C., Geoffroy, V., Maroteaux, L., Launay, J.-M., de Vernejoul, M.-C. The serotonin 5-HT_2B receptor controls bone mass via osteoblast recruitment and proliferation.