Mature osteoclasts express the 1α-hydroxylase enzyme (CYP27B1) and vitamin D receptor (VDR) and are able to synthesise and respond to 1,25(OH)₂D₃. Whether VDR signalling within osteoclasts is necessary for the regulation of osteoclastic bone resorption remains unclear. To determine the requirement for VDR-mediated activity in mature osteoclasts, a conditional deletion mouse model was created whereby VDR was deleted by breeding VDR-loxP and Cathepsin K-Cre mice (Ctsk^Cre/Vdr^-/-). These mice, and littermate controls (Vdr^fl/fl), were assessed under adequate dietary conditions (0.8% Ca and 0.7% Phos) and dietary calcium/phosphate restriction (0.03% Ca, 0.08% Phos). Six-week-old Ctsk^Cre/Vdr^-/- male mice demonstrated a 10% decrease in vertebral BV/TV% (P<0.05) which was associated with a modest increase in mean osteoclast size (P<0.05). However, when young male Ctsk^Cre/Vdr^-/- mice were fed a LowCaP diet, vertebral BV/TV% was markedly decreased by a further 22% compared to control mice on the same diet (P<0.05). While these data suggest that Ctsk^Cre/Vdr^-/- mice exhibit enhanced osteoclast-driven bone resorption under suboptimal dietary conditions, no differences were observed in the circulating resorption marker CTX or the numbers of TRAP+ osteoclasts in bone. Splenocyte derived osteoclasts were isolated and co-cultured with osteocyte-like MLO-Y4 cells. Ctsk^Cre/Vdr^-/- cells exhibited increased TRAP+ osteoclast numbers by Day 10 of culture. In parallel cultures, Ctsk^Cre/Vdr^-/- cells demonstrated a 1.92-fold increase in resorbed surface and a 3.91-fold increase in the mean size of Ctsk^Cre/Vdr^-/- osteoclasts (P<0.019). These data suggest that the absence of VDR in mature osteoclasts results in exacerbated bone resorption either via increased size and/or survival of osteoclasts. Thus, an intact VDR signalling mechanism in mature osteoclasts is required for the moderation of bone resorption.