G protein-coupled receptor 68 (GPR68) responds to extracellular protons, thus called the proton-sensing G protein-coupled receptor (GPCR), leading to activation of the phospholipase C-β (PLCβ)/calcium (Ca) pathway or the adenylyl cyclase (AC)/cyclic AMP (cAMP) pathway. We recently found that whole body deletion of Gpr68 (Gpr68 mice) reduced the number of B lymphocytes with age and during hematopoietic regeneration, such as in response to fluorouracil (5-FU) administration. This prompted us to characterize the hematopoietic stem cell (HSC) phenotype in Gpr68 mice. Despite high level of Gpr68 protein expression on HSC in bone marrow (BM), the pool size of HSC was unaltered in Gpr68 mice either under steady state or upon stress, including aging and 5-FU treatment. HSC from Gpr68 mice exhibited comparable cellular features, such as cell cycle quiescence and cell survival. HSC from Gpr68 mice also exhibited comparable competitiveness after serial transplantation. Surprisingly, cytosolic Ca accumulation was increased in HSC from Gpr68 mice. In contrast, cAMP levels were reduced in hematopoietic stem and progenitor cells (HSPC) from Gpr68 mice. Intriguingly, we found high level of Gpr68 protein expression on non-hematopoietic cells in BM, especially endothelial cells that function as HSC niche. In addition, expression of other proton-sensing GPCR was upregulated in HSPC from Gpr68 mice. Our studies suggest that Gpr68 mice display insignificant phenotype on HSC biology, possibly due to the function of Gpr68 in non-hematopoietic cells and/or the compensatory effects from other proton-sensing GPCR.Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.