Interactions of myeloma cells with the bone marrow microenvironment lead to enhanced osteoclast recruitment and impaired osteoblast activity. Recent evidence revealed that the proteasome inhibitor bortezomib stimulates osteoblast differentiation, but the mechanisms are not fully elucidated. We hypothesised that bortezomib could influence osteoblastic differentiation via alteration of vitamin D signalling by blocking the proteasomal degradation of the vitamin D receptor (VDR). This is of clinical importance, as a high rate of vitamin D deficiency was reported in patients with myeloma. We performed cocultures of primary human mesenchymal stem cells (hMSCs) and human osteoblasts (hOBs) with myeloma cells, which resulted in an inhibition of the vitamin D-dependent differentiation of osteoblast precursors. Treatment with bortezomib led to a moderate increase in osteoblastic differentiation markers in hMSCs and hOBs. Importantly, this effect could be strikingly increased when vitamin D was added. Bortezomib led to enhanced nuclear VDR protein levels in hMSCs. Primary hMSCs transfected with a VDR luciferase reporter construct showed a strong increase in VDR signalling with bortezomib. In summary, stimulation of VDR signalling is a mechanism for the bortezomib-induced stimulation of osteoblastic differentiation. The data suggest that supplementation of vitamin D in patients with myeloma treated with bortezomib is crucial for optimal bone formation.