Hematopoietic stem cell (HSC) transplantation (HSCT) represents the only curative therapy for most hematologic disease including leukemia. Recent studies suggest that “stress hematopoiesis”, including that which occurs post-HSCT, is subject to distinct regulation compared steady-state hematopoiesis. Better understanding the molecular mechanisms that regulate transplanted HSCs and their engraftment is necessary to improve HSCT. Thus, we seek to identify novel molecular regulators of HSCT. Our data reveal high expression of multiple GPRASPs (G-Protein Coupled Receptor Associated Sorting Proteins) (e.g. Gprasp1 and Gprasp2) in HSCs relative to downstream progenitors. ShRNA-mediated gene knock-down transplantation-based studies revealed that both GPRASP1 and GPRASP2 act as negative regulators of HSCT, showing 4-fold increase in peripheral blood and bone marrow reconstitution and significantly enhance survival and quiescence in HSCs ex vivo and after transplant. GPRASPs regulate the post-endosomal trafficking of GPCRs to the lysosome. CXCR4, a GPCR that regulates HSCs function, contains a putative GPRASP-binding motif in its C-terminus, implicating it as a possible target of GPRASP-mediated degradation. Indeed, Gprasp1 or Gprasp2 loss increased both total CXCR4 and cell membrane-localized CXCR4 in HSPCs. Consequently, Gprasp1 or Gprasp2 loss increased HSPC migration toward SDF-1. Further, Gprasp1 or Gprasp2 loss significantly enhanced acute homing and HSCs niche retention post-transplant. These GPRASP-knockdown effects disappear when Cxcr4 is genetically deleted. In sum, we report for the first time that multiple GPRASPs function as barriers to HSCT by effecting HSC migration, homing and niche retention via regulation of CXCR4 stability and localization.