This study examined the effect of isoflavone-enriched milk on bone loss in ovariectomized (OVX) rats. Thirty 6-week-old Sprague-Dawley female rats were divided into two groups: sham-operated and OVX. The OVX group was subdivided into three dietary groups (OVX, non-isoflavone-enriched milk; OVX+Iso, isoflavone-enriched milk; and OVX+Iso+Vit+Ca, isoflavone-, vitamins D and K-, and Ca-enriched mik). After 19 weeks of feeding, the food efficiency ratio and body weight gain in the sham-operated group were significantly lower compared with those in the other groups. The bone alkaline phosphatase and total alkaline phosphatase activities were significantly higher in isoflavone-enriched groups (OVX+Iso and OVX+Iso+Vit+Ca) when compared with the sham-operated group. Urinary excretions of deoxypyridinoline and hydroxyproline were significantly higher with ovariectomy, but mostly normalized in the OVX+Iso and OVX+Iso+Vit+Ca groups. The rats in the OVX+Iso and OVX+Iso+Vit+Ca groups showed higher femur and tibia weights. A significant increase was found in bone density of femur and trabecular bone area in the OVX+Iso+Vit+Ca group, which almost reached that of the sham-operated group, whereas no difference was found among the OVX and OVX+Iso groups. The histological areas of the proximal tibia sections showed highly filled trabecular bone in both isoflavone-enriched groups (OVX+Iso and OVX+Iso+Vit+Ca). The present study indicated that isoflavone-enriched milk may have a partial preventive effect on ovariectomy-induced bone loss; however, vitamins D and K and Ca enrichment with isoflavone may enhance effectiveness for increasing bone mass in OVX rats.