Identification of cues from skeletal muscle that control bone formation is essential for understanding the crosstalk between muscle and bone and for developing novel therapeutics for degenerative bone diseases. Here, we identified that skeletal muscle secreted abundant extracellular vesicles (Mu-EVs). Mu-EVs traveled via blood vessels to bone and were phagocytized by bone marrow mesenchymal stem/stromal cells (BMSCs). Mu-EVs promoted osteogenic differentiation of BMSCs and protected mice against disuse osteoporosis. The quantity and bioactivity of Mu-EVs were tightly correlated with the function of skeletal muscle. Protein mass spectrometry revealed dozens of proteins in Mu-EVs potentially involved in bone metabolism regulation, especially glycolysis. Further analysis indicated that Mu-EVs promoted the glycolysis of BMSCs by delivering LDHA into BMSCs to promote glycolysis. Taken together, these findings uncovered that Mu-EVs function as a non-canonical myokine to regulate BMSC metabolism and bone formation. Supplementation of normal Mu-EVs is a potential therapy for disuse osteoporosis.