O variectomy in young, growing rodents results in decreased trabecular bone mineral density (BMD) and increased radial growth of the cortical bone. Both of these effects are reversed by treatment with estrogen. The aim of the present study was to determine the physiological role of estrogen receptor-β (ERβ) on bone structure and bone mineral content (BMC). The BMC was increased in adult (11 weeks old), but not prepubertal (4 weeks old), female ERβ^–/– mice compared with wild-type (WT) mice. This increase in BMC in females was not due to increased trabecular BMD, but to an increased cross-sectional cortical bone area associated with a radial bone growth. Male ERβ^–/– mice displayed no bone abnormalities compared with WT mice. Ovariectomy decreased the trabecular BMD to the same extent in adult female ERβ^–/– mice as in WT mice. The expression levels of osteoblast-associated genes — α1(I) collagen, alkaline phosphatase, and osteocalcin mRNAs — were elevated in bone from adult ERβ^–/– females compared with WT mice. These observations provide a possible explanation for the increased radial bone growth seen in female mutants, suggesting a repressive function for ERβ in the regulation of bone growth during female adolescence. In summary, ERβ is essential for the pubertal feminization of the cortical bone in female mice but is not required for the protective effect of estrogens on trabecular BMD.