Fructooligosaccharides (FOS) stimulate the growth of bifidobacteria, which cleave isoflavone conjugates to yield the corresponding aglycones and metabolites. In a previous study, FOS modified the absorption and enterohepatic recirculation of isoflavones in rats. In the present study, we determined the effect of the combination of dietary FOS and isoflavone conjugates on bone mass in ovariectomized (OVX) and surgical control mice. After undergoing OVX or sham operation, female ddY mice (8 wk old, n = 64) were randomly assigned to four groups: a purified control diet (AIN-93G) group, a FOS diet (AIN-93G + 5% FOS) group, an isoflavone diet (AIN-93G + 0.2% isoflavone conjugates) group, or a FOS and isoflavone diet (AIN-93G + 5% FOS + 0.2% isoflavone conjugates) group. After 6 wk, the mice were killed and the blood and femora were sampled immediately. In OVX mice, both isoflavone conjugates and FOS prevented femoral bone loss. An additive effect of dietary isoflavone conjugates and FOS was observed by dual-energy X-ray absorptiometry in the distal part of the femur and in trabecular bone, by peripheral quantitative computed tomography. Moreover, FOS increased cecal beta-glucosidase activity and equol production from daidzein in both OVX and surgical control mice fed isoflavone conjugates. These results suggest that FOS increase the bioavailability of isoflavones, leading to cooperative effects in the prevention of osteopenia in OVX mice.