We examined whether estrogen receptor (ER)α is required for estrogen to stimulate cancellous bone formation in long bones of male mice. 17β-Estradiol (E₂) was administered to ERα^−/− male mice or wild-type (WT) littermate controls at 40, 400, or 4000 μg/kg by daily sc injection for 28 d and histomorphometric analysis performed at the distal femoral metaphysis. In WT mice, treatment with E₂ (40 μg/kg·d) increased the proportion of cancellous bone surfaces undergoing mineralization and stimulated mineral apposition rate. In addition, higher doses of E₂ induced the formation of new cancellous bone formation surfaces in WT mice. In contrast, E₂ had little effect on any of these parameters in ERα^−/− mice. Immunohistochemistry was subsequently performed using an ERα-specific C-terminal polyclonal antibody. In WT mice, ERα was expressed both by cancellous osteoblasts and a significant proportion of mononuclear bone marrow cells. Immunoreactivity was also observed in cancellous osteoblasts of ERα^−/− mice, resulting from expression of the activation function-1-deficient 46-kDa ERα isoform previously reported to be expressed in normal osteoblasts and bones of ERα^−/− mice. Taken together, our results suggest that estrogen stimulates bone formation in mouse long bones via a mechanism that requires the presence of full-length ERα possessing activation function-1.