The L-gulono-γ-lactone oxidase gene ( Gulo ) encodes an essential enzyme in the synthesis of ascorbic acid from glucose. On the basis of previous findings of bone abnormalities in Gulo ^-/- mice under conditions of ascorbic acid insufficiency, we investigated the effect of ascorbic acid insufficiency on factors related to bone metabolism in Gulo ^-/- mice. Four groups of mice were raised for 4 weeks under differing conditions of ascorbic acid insufficiency, namely, wild type; ascorbic acid-sufficient Gulo ^-/- mice, 3-week ascorbic acid-insufficient Gulo ^-/- mice, and 4-week ascorbic acid-insufficient Gulo ^-/- mice. Four weeks of ascorbic acid insufficiency resulted in significant weight loss in Gulo ^-/- mice. Interestingly, average plasma osteocalcin levels were significantly decreased in Gulo ^-/- mice after 3 weeks of ascorbic acid insufficiency. In addition, the tibia weight in ascorbic acid-sufficient Gulo ^-/- mice was significantly higher than that in the other three groups. Moreover, significant decreases in trabecular bone volume near to the growth plate, as well as in trabecular bone attachment to the growth plate, were evident in 3- or 4-week ascorbic acid-insufficient Gulo ^-/-. In summary, ascorbic acid insufficiency in Gulo ^-/- mice results in severe defects in normal bone formation, which are closely related to a decrease in plasma osteocalcin levels.