A cell-free approach utilizing the paracrine effects of mesenchymal stromal cells is receiving attention in regenerative medicine. In the present study, we evaluated the effects of a conditioned medium of amniotic fluid-derived stromal cells (AFSC-CM) on bone metabolism. In mice, intraperitoneal injections of AFSC-CM increased bone mass and enhanced bone turnover. The precursor populations of myeloid and mesenchymal lineages, as well as endothelial cells in bone marrow, were also augmented by AFSC-CM administration. In an in vitro culture experiment, AFSC-CM increased osteoclast differentiation of bone marrow-derived macrophages, but had no significant effect on the osteogenic differentiation of preosteoblasts. However, AFSC-CM administration dramatically accelerated the migration and tube formation of endothelial cells, and a cytokine array showed that AFSC-CM contained many angiogenic factors. These results indicate that AFSC-CM exerts a bone anabolic effect by changing the bone marrow microenvironment, including angiogenesis and precursor expansion. Therefore, ameliorating marrow angiogenesis is a potential therapeutic strategy for bone regeneration, for which AFSCs can be a good cellular source.