Restenosis remains the main reason for treatment failure of arterial disease. Sirolimus (SIR) as a potent anti-proliferative agent is believed to prevent the phenomenon. The application of exosomes provides an extended-release delivery platform for SIR intramural administration. Herein, SIR was loaded into fibroblast-derived exosomes isolated by ultracentrifugation. Different parameters affecting drug loading were optimized, and exosome samples were characterized regarding physicochemical, pharmaceutical, and biological properties. Cytotoxicity, scratch wound assays, and quantitative real-time PCR for inflammation- and migration-associated genes were performed. Restenosis was induced by carotid injury in a rat carotid model and then exosomes were locally administered. After 14 days, animals were investigated by computed tomography (CT) angiography, morphometric, and immunohistochemical analyses. Western blotting confirmed the presence of specific protein markers in exosomes. Characterization of empty and SIR-loaded exosomes verified round and nanoscale structure of vesicles. Among prepared formulations, desired entrapment efficiency (EE) of 76% was achieved by protein:drug proportion of 2:1 and simple incubation for 30 min at 37 °C. Also, the optimal formulation released about 30% of the drug content during the first 24 h, followed by a prolonged release for several days. In vitro studies revealed the uptake and functional efficacy of the optimized formulation. In vivo studies revealed that %restenosis was in the following order: saline > empty exosomes > SIR-loaded exosomes. Furthermore, Ki67, alpha smooth muscle actin (α-SMA), and matrix metalloproteinase (MMP) markers were less expressed in the SIR-exosomes-treated arteries. These findings confirmed that exosomal SIR could be a hopeful strategy for the prevention of restenosis.