We have reported that soy isoflavones are capable of preventing loss of bone mineral density (BMD) in rats due to ovariectomy. The intestinal microflora is important in rendering soy isoflavones bioavailability by facilitating their conversion to equol. Hence, substances that can modulate the intestinal microflora could affect the bioavailability of isoflavones. The purpose of this study was to examine whether combination of genistin and fructooligosaccharides (FOS), a prebiotic, can enhance the effects of soy isoflavones on bone in ovariectomized (OVX) female rats. Forty-eight 90-day-old female Sprague-Dawley rats were either sham-operated (Sham; one group) or Ovx (three groups) and were placed on dietary treatment for 50 days. The Sham and one Ovx group received a control diet, and the remaining Ovx groups received genistin-rich isoflavones diet (Ovx+G) or genistin-rich isoflavones and FOS diet (Ovx+G+FOS). After 50 days, blood and bone specimens were collected for analysis. The genistin-rich isoflavones diet was able to significantly increase the whole-body, right femur, and fourth lumbar BMD by 1.6%, 1.48%, and 1.3%, respectively in comparison with the Ovx control. The combination of genistin-rich isoflavones diet and 5% FOS further increased whole-body, right femur, and fourth lumbar BMD more compared to the genistin-rich isoflavones diet. Our findings suggest that although a genistin-rich isoflavones diet can increase the BMD in rats with Ovx-induced bone loss, combination of genistin-rich isoflavones and FOS had greater effect in preventing bone loss in this rat model.