We evaluated the protective effects of soy isoflavones (SIF) against osteoporosis in middle-aged ovariectomized (OVX) mice. SIF (30 mg/kg or 60 mg/kg) or 17beta-estradiol (E(2)) was administered to OVX mice for 4 months after bilateral ovariectomy. We observed the biochemical markers of bone turnover, e.g., alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), in serum. We also observed the bone mineral density (BMD) in femurs and lumbar vertebrae. In addition, we examined trabecular bone and interstitial cells in the femur using hematoxylin and eosin staining. The decrease in ALP levels and the increase in TRAP levels normally resulting from ovariectomy were suppressed by administration of 60 mg/kg SIF or E(2). Administration of 60 mg/kg SIF or E(2) also maintained the BMD, trabecular bone, and interstitial cells in OVX mice compared to those in pre-OVX mice. These results suggest that 60 mg/kg SIF effectively mitigates ovariectomy-induced osteoporosis in middle-aged mice.