Mesenchymal stem cells (MSCs) reside in a perivascular space surrounding bone marrow sinusoids and provide the microenvironment for hematopoietic progenitor cells. However, fundamental properties of perisinusoidal MSCs in their native environment remain elusive. A single cell RNA-seq analysis of marrow stromal cells expressing C-X-C motif chemokine 12 (CXCL12)-GFP revealed functionally distinct cellular subsets; we found a perisinusoidal subpopulation expressing abundant secreted niche factors was preferentially marked by Cxcl12-creER upon tamoxifen injection. Further in vivo cell lineage analysis revealed that these perisinusoidal MSCs remained unexpectedly dormant in physiological conditions without generating new reticular cells during marrow growth, but became rapidly activated in response to injury and effectively regenerated bones. These findings unravel a uniquely bimodal functionality of perisinusoidal MSCs, highlighting their characteristics as reserve stem cells that are dedicated to supporting hematopoiesis in homeostasis but can be enlisted to provide a significant cellular source during regeneration of the skeletal organ.