A putative involvement of the vasculature seems to play a critical role in the pathophysiology of graft-versus-host disease (GVHD). We aimed to characterize alterations of mesenteric resistance arteries in GVHD in a fully MHC-mismatched model of BALB/c mice conditioned with total body irradiation that underwent transplantation with bone marrow cells and splenocytes from syngeneic (BALB/c) or allogeneic (C57BL/6) donors. After 4 weeks, animals were sacrificed and mesenteric resistance arteries were studied in a pressurized myograph. The expression of endothelial (eNOS) and inducible nitric oxide (NO)-synthase (iNOS) was quantified and vessel wall ultrastructure was investigated with electron microscopy. The myograph study revealed an endothelial dysfunction in allogeneic-transplant recipients, whereas endothelium-independent vasodilation was similar to syngeneic-transplant recipients or untreated controls. The expression of eNOS was decreased and iNOS increased, possibly contributing to endothelial dysfunction. Additionally, arteries of allogeneic transplant recipients exhibited a geometry-independent increase in vessels strain. For both findings, electron microscopy provided a structural correlate by showing severe damage of the whole vessel wall in allogeneic-transplant recipient animals. Our study provides further data to prove, and is the first to characterize, functional and structural vascular alterations in the early course after allogeneic transplantation directly in an ex vivo setting and, therefore, strongly supports the hypothesis of a vascular form of GVHD.