Wnt1 is one of the several glycoproteins activating Wnt signaling, critical for normal skeletal development and bone homeostasis. Wnt1 was previously believed to solely regulate central nervous system development, in particular in midbrain and cerebellum. However, remarkable findings have recently shown that several patients affected by severe form of Osteogenesis Imperfecta (OI) display a Wnt1 mutation thereby revealing a possible role of Wnt1 in bone metabolism. Here, we show that recombinant Wnt1 (r-Wnt1) strongly increases differentiation of bone marrow stromal cells into mature osteoblasts, as demonstrated by the enhanced number of cells positively stained for alkaline phosphatase, one of the osteoblastic marker genes, whose mRNA levels are also significantly up-regulated. Furthermore, other osteogenic master genes such as Collagen I and Osteopontin are also enhanced when bone marrow precursors were differentiated toward osteoblastic phenotype in the presence of r-Wnt1. Intriguingly, by in vivo and in vitro findings, we report that in the bone marrow of mice subjected to physical activity there is a high endogenous Wnt1 synthesis compared to mice kept in resting conditions. Moreover, conditioned medium collected from ex vivo myoblasts, harvested from exercised mice, up-regulates Wnt1 expression in osteoblast cell cultures obtained from control mice. Overall our findings support the role of Wnt1 in regulating bone metabolism and suggest that this molecule could be one of the mediators through which physical activity may exert beneficial effect on bone.