Gaseous signaling molecules such as hydrogen sulfide (H₂S) are produced endogenously and mediate effects through diverse mechanisms. H₂S is one such gasotransmitters that regulates multiple signaling pathways in mammalian cells, and abnormal H₂S metabolism has been linked to defects in bone homeostasis. Here, we demonstrate that bone marrow mesenchymal stem cells (BMMSCs) produce H₂S in order to regulate their self-renewal and osteogenic differentiation, and H₂S deficiency results in defects in BMMSC differentiation. H₂S deficiency causes aberrant intracellular Ca²⁺ influx because of reduced sulfhydration of cysteine residues on multiple Ca²⁺ TRP channels. This decreased Ca²⁺ flux downregulates PKC/Erk-mediated Wnt/β-catenin signaling which controls osteogenic differentiation of BMMSCs. Consistently, H₂S-deficient mice display an osteoporotic phenotype that can be rescued by small molecules that release H₂S. These results demonstrate that H₂S regulates BMMSCs and that restoring H₂S levels via nontoxic donors may provide treatments for diseases such as osteoporosis that can arise from H₂S deficiencies.