Vitamin K(2) (K(2), menatetrenone) has been reported to enhance bone formation and inhibit bone resorption in vitro. However, there is no evidence that K(2) enhances bone formation in vivo. The aim of this study was to characterize the effect of K(2) on bone formation in vivo. We carried out two experiments using a prednisolone (pred)-induced bone loss model in male (10-week-old) Fischer rats. Pred was orally administered three times a week. In experiment 1, we compared the degree of bone loss induced by a 4 week treatment (30 or 100 mg/kg) and an 8 week treatment (3, 10, or 30 mg/kg) with pred by peripheral quantitative computed tomography (pQCT). At 4 weeks, total bone mineral density (BMD) was decreased only with the 100 mg/kg pred treatment. At 8 weeks, total BMD was significantly reduced at >10 mg/kg pred. In experiment 2, we investigated the effect of K(2) on bone loss induced by 3 and 30 mg/kg pred. K(2) (15 mg/kg) was given to rats as a dietary supplement for 8 weeks. Intestinal calcium transport (S/M) and total, trabecular, and cortical BMD at the metaphysis and diaphysis were measured, and histomorphometry was performed in diaphysial cross sections. Pred treatment decreased total and trabecular BMD in the proximal metaphysis. A decrease in cortical BMD in the diaphysis was observed in the pred 30 mg/kg group. Pred treatment also reduced mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS). The decrease in total and trabecular BMD in the proximal metaphysis, and in cortical BMD in the diaphysis, was inhibited by K(2) treatment. K(2) treatment also inhibited the decrease in MS/BS and BFR/BS induced by 30 mg/kg pred. These results suggest that K(2) prevents bone loss partly through the enhancement of bone formation.