Vitamin D depletion and low dietary levels of calcium can independently cause bone loss in rodents consistent with osteoporosis. Although high levels of dietary calcium can normalise bone volume independently of vitamin D activity, little consideration has been given to the quality of the bone mineral that is formed in the absence of sufficient vitamin D, as determined by 25-hydroxyvitamin D (25D) levels. To investigate the role of dietary calcium and vitamin D on bone material properties, 3 month old female Sprague-Dawley rats (n=5-6/grp) were pair-fed a semi-synthetic diet containing either 0 (LD) or 1000IU/kg (HD) vitamin D with either low (0.1%, LC) or high (1%, HC) dietary calcium for 3 months. At 6 months of age, animals were killed to measure blood biochemistry, and femoral histomorphometric and sub-structural bone properties. LD fed mice reduced serum 25D levels approximately 6-fold relative to HD mice. Serum calcium levels were normal and not different between groups. In LC/LD fed mice, the proximal femoral bone mineral volume (BV/TV) was approximately 30% lower than HC/LD or HD mice (P<0.001), indicating that HC is sufficient to normalise bone volume independent of vitamin D status. However, calcium and calcium-phosphate concentration within the bone were both diminished by approximately 30% in LD mice even when fed the HCa diet. Consistent with this, the hydroxyproline levels were reduced by 35% (P<0.001) when compared with HD mice, suggesting compromised collagen stability in LD mice regardless of dietary calcium. These data indicate that while adequate dietary calcium is capable of normalising bone volume, the absence of sufficient 25D impairs collagen stability and calcium density within the bone micro-environment.