We recently found that estrogen receptor (ER) antagonists prevent high-dose estrogen from inducing the formation of new cancellous bone within the medullary cavity of mouse long bones. In the present investigation, we studied the role of specific ER subtypes in this response by examining whether this is impaired in female ERβ^−/− mice previously generated by targeted gene deletion. Vehicle or 17β-estradiol (E₂) (range 4–4,000 μg · kg⁻¹ · day⁻¹) was administered to intact female ERβ^−/− mice and wild-type littermates by subcutaneous injection for 28 days. The osteogenic response was subsequently assessed by histomorphometry performed on longitudinal and cross sections of the tibia. E₂ was found to cause an equivalent increase in cancellous bone formation in ERβ^−/− mice and littermate controls, as assessed at the proximal and distal regions of the proximal tibial metaphysis. E₂ also resulted in a similar increase in endosteal mineral apposition rate in these two genotypes, as assessed at the tibial diaphysis. In contrast, cortical area in ERβ^−/− mice was found to be greater than that in wild types irrespective of E₂ treatment, as was tibial bone mineral density as measured by dual-energy X-ray absorptiometry, consistent with previous reports of increased cortical bone mass in these animals. We conclude that, although ERβ acts as a negative modulator of cortical modeling, this isoform does not appear to contribute to high-dose estrogen's ability to induce new cancellous bone formation in mouse long bones.