Osteoporosis and muscle frailty are important health problems in elderly men and may be partly related to biological androgen activity. This androgen action can be mediated directly through stimulation of the androgen receptor (AR) or indirectly through stimulation of estrogen receptor‐alpha (ERα) following aromatization of androgens into estrogens. To assess the differential action of AR and ERα pathways on bone and body composition, AR‐ERα double‐knockout mice were gener‐ated and characterized. AR disruption decreased trabec‐ular bone mass, whereas ERα disruption had no additional effect on the AR‐dependent trabecular bone loss. In contrast, combined AR and ERα inactivation additionally reduced cortical bone and muscle mass compared with either AR or ERα disruption alone. ERα inactivation—in the presence or absence of AR—increased fat mass. We demonstrate that AR activation is solely responsible for the development and maintenance of male trabecular bone mass. Both AR and ERα activation, however, are needed to optimize the acquisition of cortical bone and muscle mass. ERα activation alone is sufficient for the regulation of fat mass. Our findings clearly define the relative importance of AR and ERα signaling on trabecu‐lar and cortical bone mass as well as body composition in male mice.—Callewaert, F., Venken, K., Ophoff, J., De Gendt, K., Torcasio, A., van Lenthe, G. H., Van Ooster‐wyck, H., Boonen, S., Bouillon, R., Verhoeven, G., Vanderschueren, D. Differential regulation of bone and body composition in male mice with combined inactivation of androgen and estrogen receptor‐α. FASEB J. 23, 232‐240 (2009)