Endothelial cells (ECs) are components of the hematopoietic microenvironment and regulate hematopoietic stem and progenitor cell (HSPC) homeostasis. Cytokine treatments that cause HSPC trafficking to peripheral blood are associated with an increase in dipeptidylpeptidase 4/CD26 (DPP4/CD26), an enzyme that truncates the neurotransmitter neuropeptide Y (NPY). Here, we show that enzymatically altered NPY signaling in ECs caused reduced VE-cadherin and CD31 expression along EC junctions, resulting in increased vascular permeability and HSPC egress. Moreover, selective NPY2 and NPY5 receptor antagonists restored vascular integrity and limited HSPC mobilization, demonstrating that the enzymatically controlled vascular gateway specifically opens by cleavage of NPY by CD26 signaling via NPY2 and NPY5 receptors. Mice lacking CD26 or NPY exhibited impaired HSPC trafficking that was restored by treatment with truncated NPY. Thus, our results point to ECs as gatekeepers of HSPC trafficking and identify a CD26-mediated NPY axis that has potential as a pharmacologic target to regulate hematopoietic trafficking in homeostatic and stress conditions.