Mesenchymal stem cell (MSC)-mediated bone regeneration is used to replace lost bone. However, methods to accelerate the process and stabilize regenerated bone remain limited. Therefore, we investigated the effect of bisphosphonates (BPs) on the function of bone marrow mesenchymal stem cells (BMMSCs) to determine if they might enhance MSC-mediated bone regeneration. We isolated and cultured BMMSCs from BALB/c mice and treated the cells with 0.1, 0.5, 1, 5, or 10 mu M zoledronic acid (ZA; Zometa, a commercially available BP). ZA had a dose-dependent effect on BMMSCs proliferation and osteogenesis. ZA at concentrations of 5 and 10 mu M inhibited the proliferation and osteogenic differentiation of BMMSCs. By contrast, in addition to inducing the proliferation and osteogenesis of BMMSCs, 0.5 mu M ZA upregulated expressions of the osteogenesis-related genes Alp, osterix (Osx), and bone sialoprotein (Bsp) and enhanced osteogenesis in vivo when ZA-treated BMMSCs were implanted subcutaneously in nude mice. In addition, 0.5 mu M ZA increased expression of Opg in BMMSCs, decreased the Rankl/Opg ratio, and decreased the number of osteoclasts. However, it was not associated with adverse effects on numbers of regulatory T cells or levels of Th17, transforming growth factor-beta 1 (TGF-beta 1), and interleukin-17a (IL-17a) when cocultured with T cells. In conclusion, 0.5 mu M ZA pretreatment enhanced the proliferation and osteogenesis of BMMSCs in vitro and in vivo and decreased the number of osteoclasts without impairment of BMMSCs immunomodulatory properties. In vitro pretreatment of BMMSCs with BP and subsequent implantation may be a safe and effective way of enhancing MSC-mediated bone regeneration.