Vitamin D metabolites such as 25-hydroxyvitamin D (25D) circulate bound primarily to vitamin D binding protein (DBP). However, for most extra-renal tissues 25D uptake is independent of DBP, even though the free 25D fraction is very small. DBP has a lower binding affinity for 25D2 compared to 25D3. We hypothesized that this would increase serum free 25D2, with possible variations in vitamin D function. Mice were placed on diets containing equal amounts (1000 IU/kg) of vitamin D2 or D3 at week 3 of age. At week 8 mice fed D2 diet had only 25D2 in circulation (26.6 ng/ml±1.9), and mice fed D3 mice had only 25D3 (28.3 ng/ml±2.0). By contrast, measured free 25D was significantly higher in D2 animals (16.8 pg/ml±0.65 vs 8.4 pg/ml±0.63, P<0.001). Parathyroid hormone showed no significant difference between D2 and D3 mice (193 pg/ml±9.0 vs 196 pg/ml±6.2). However, analysis of spleens from week 8 D2 and D3 mice showed that in female mice on D2 there was increased mRNA expression of the vitamin D-activation enzyme Cyp27b1 (2.35-fold±0.45), the monocyte-macrophages marker CD11b (1.77-fold±0.45), and the osteoclastogenesis precursor RANKL (1.80-fold±0.37) relative to female D3 mice. Conversely, another monocyte marker, CD14, showed decreased mRNA expression (0.39-fold±0.19) in D2 vs D3 mice. Flow cytometry revealed a significant increase in total CD45+ monocyte-macrophage populations in spleens from D2 females (38.04%) compared to D3 females (P=0.0173). Conversely natural killer cells were reduced in D2 mice (39.25%) compared to D3 mice (P=0.0054). There was no difference in T cell or B cell markers between D2 and D3 mice. These data suggest that free 25D (25D2>25D3) has extra skeletal effects that include effects upon immune cell development.