Bone homeostasis is achieved by the balance between osteoclast-dependent bone resorption and osteoblastic events involving differentiation of adult mesenchymal stem cells (MSCs). Prostate carcinoma (PC) cells display the propensity to metastasize to bone marrow where they disrupt bone homeostasis as a result of mixed osteolytic and osteoblastic lesions. The PC-dependent activation of osteoclasts represents the initial step of tumor engraftment into bone, followed by an accelerated osteoblastic activity and exaggerated bone formation. However, the interactions between PC cells and MSCs and their participation in the disease progression remain as yet unclear. In this study, we show that bone metastatic PC-3 carcinoma cells release factors that increase the expression by human (h)MSCs of several known pro-osteoblastic commitment factors, such as α5/β1 integrins, fibronectin, and osteoprotegerin. As a consequence, as shown in an osteogenesis assay, hMSCs treated with conditioned medium (C(ed) M) derived from PC-3 cells have an enhanced potential to differentiate into osteoblasts, as compared to hMSCs treated with control medium or with C(ed) M from non-metastatic 22RV1 cells. We demonstrate that FGF-9, one of the factors produced by PC-3 cells, is involved in this process. Furthermore, we show that PC-3 C(ed) M decreases the pro-osteoclastic activity of hMSCs. Altogether, these findings allow us to propose clues to understand the mechanisms by which PC favors bone synthesis by regulating MSC outcome and properties.