Abstract Macrophages, play important roles in initiation and resolution of inflammation. Notably, a subset of tissue macrophages strategically localizes to the blood vessels, exerting essential regulatory functions at the blood-tissue interface, including regulation of vascular permeability and neutrophil (PMN) transendothelial migration (TEM). Although the number of vessel-associated macrophages (VAMs) is increased in inflamed tissue, the underlying mechanisms of VAMs recruitment are unknown. Our current study uncovers a novel mechanism whereby inflamed intestinal vascular endothelial cells (ECs) release IL-16 to selectively recruit CD4+ tissue macrophages to become VAMs. Whole-mount confocal microscopy studies in CX3CR1-EGFP macrophages reporter mice confirmed significant increases in VAMs numbers in several murine models of intestinal inflammation. Surprisingly, the majority of VAMs were found to express a classical T cell marker, CD4 and to be of an inflammatory phenotype, enriched for ICAM-1 and TNFα expression. VAMs-derived TNFα was previously found to prime ECs to form PMN TEM hot spots. Whole genome mRNA sequencing, and flow cytometry protein expression analyses revealed heightened expression of the CD4 ligand, IL-16 by inflamed intestinal lamina propria ECs. In vitro, using transwell assays, recombinant IL-16 and supernatants derived from murine (bEnds.3) endothelial cells induced robust migration of murine, LPS/IFNγ-stimulated bone marrow-derived macrophages. Whereas antibody-mediated IL-16 or CD4 neutralization in cultures and in vivo reduced VAMs recruitment. Collectively, our findings establish CD4 as a novel marker of VAMs and identify the regulatory role of ECs IL-16 and macrophages CD4+ axis in VAMs recruitment in inflamed mucosal tissues.