Cross-sex hormone therapy (XHT) is widely used by transgender people to alter secondary sex characteristics to match their desired gender presentation. Here, we investigate the long-term effects of XHT on bone health using a murine model. Female mice underwent ovariectomy at either 6 or 10 wk and began weekly testosterone or vehicle injections. Dual-energy X-ray absorptiometry (DXA) was performed (20 wk) to measure bone mineral density (BMD), and microcomputed tomography was performed to compare femoral cortical and trabecular bone architecture. The 6-wk testosterone group had comparable BMD with controls by DXA but reduced bone volume fraction, trabecular number, and cortical area fraction and increased trabecular separation by microcomputed tomography. Ten-week ovariectomy/XHT maintained microarchitecture, suggesting that estrogen is critical for bone acquisition during adolescence and that late, but not early, estrogen loss can be sufficiently replaced by testosterone alone. Given these findings, we then compared effects of testosterone with effects of weekly estrogen or combined testosterone/low-dose estrogen treatment after a 6-wk ovariectomy. Estrogen treatment increased spine BMD and microarchitecture, including bone volume fraction, trabecular number, trabecular thickness, and connectivity density, and decreased trabecular separation. Combined testosterone-estrogen therapy caused similar increases in femur and spine BMD and improved architecture (increased bone volume fraction, trabecular number, trabecular thickness, and connectivity density) to estrogen therapy and were superior compared with mice treated with testosterone only. These results demonstrate estradiol is critical for bone acquisition and suggest a new cross-sex hormone therapy adding estrogens to testosterone treatments with potential future clinical implications for treating transgender youth or men with estrogen deficiency.