Extracellular vesicles (EVs) have become a key tool in the biotechnological landscape due to their well-documented ability of mediating intercellular communication. Such feature has been explored and, actually, it is under constant investigation by researchers, who have unraveled the important role of EVs in several research fields ranging from oncology, immunology and diagnostics to regenerative medicine. Unfortunately, there are still some limits to overcome before a clinical application, including the inability to confine the EVs to strategically defined sites of interest, to avoid side effects. In this study, for the first time, EVs application is supported by 3D bioprinting technology to develop a new strategy for applying the angiogenic cargo of HUVEC-derived EVs in regenerative medicine. EVs, derived from human endothelial cells and grown under different stressed conditions, were collected and used as bio-additives for the formulation of advanced bioinks. After in vivo sub-cutaneous implantation, we demonstrated that the bioprinted 3D structures, loaded with EVs, supported the formation of a new functional vasculature in situ, consisting of blood-perfused microvessels recapitulating the printed pattern. The results obtained in this study favor the development of new therapeutic approaches for critical clinical conditions, such as the need for prompt revascularization of ischemic tissues, which represents the fundamental substrate for advanced regenerative medicine applications.Creative Commons Attribution license.