The aim of the present study was to evaluate the effect of dietary lactoferrin on bone metabolism in vivo using a postmenopausal animal model. We investigated whether bovine lactoferrin (bLF) ingestion could prevent bone loss in ovariectomized mice. Twelve-week-old female C3H mice either ovariectomized or sham-operated were fed for 27 weeks with the control diet (AIN-93M with 140 g of total milk protein as a protein source per kg of diet). Four group of ovariectomized mice received diets including different concentrations of bLF (1, 5, 10 or 20 g of total milk protein were replaced by bLF). Ovariectomy induced a decrease uterine weight and a smaller gain of bone mineral density. Immunoreactive bLF was detected in the peripheral blood and its concentration was related to the amount of bLF ingestion. bLF supplementation to the diet improved bone mineral density (BMD) and femoral failure load in a dose-dependent manner. We confirmed the direct effects of bLF in vitro using established and primary cultures of murine bone cells. Addition of bLF to the culture medium at concentration between 1 and 1000 µg/ml stimulated both cell growth and differentiation of osteoblastic MC3T3 cells and on the contrary, inhibited the growth of pre-osteoclastic RAW 267.4 cells. In primary culture of mixed bone cells, an enhanced osteoblast differentiation was associated with an inhibition of osteoclasts differentiation at lower bLF concentrations (1-10 µg/ml). In conclusion, these findings suggest that dietary lactoferrin supplementation have a beneficial effect on postmenopausal bone loss by modulating bone formation and resorption.