Converging lines of evidence suggest that oxidized lipids, long recognized as a risk factor in atherogenesis, also contribute to osteoporosis, but the underlying mechanism is not understood in detail. The effect of atherogenesis related factors including oxysterols on the differentiation and survival of marrow stromal cells (MSCs) would be very important in understanding the link between atherosclerosis and osteoporosis. In the present study, the effect of oxysterol cholestane-3β,5α,6β-triol (Triol) on osteoblastic differentiation and apoptosis of primary rat bone MSCs as well as the related mechanisms were studied. Triol inhibited MSCs osteoblastic differentiation as demonstrated by inhibition of alkaline phosphatase activity, osteocalcin secretion, and matrix mineralization. In the other aspect, Triol promoted MSCs apoptosis, as characterized by condensed or fragmented nuclei as well as active externalization of phosphatidyl serine to the cell surface. In addition, Triol was found to induce increases of intracellular Ca2+ and Ca2+-dependent reactive oxygen species generation in MSCs. These effects were involved in the action of Triol on apoptosis, but not on osteoblastic differentiation of MSCs. These results suggested that Triol might contribute to the decreased bone formation by inhibition of osteoblastic differentiation and promotion of apoptosis of MSCs, providing insights about common factors underlying the pathogenesis of atherosclerosis and osteoporosis. © 2005 Wiley-Liss, Inc.