Abstract: Objective To extract and identify semen-derived exosome using PEG6000. Methods Exosomes were extracted from semen specimens from 6 healthy volunteers with step-by-step centrifugations and ultracentrifugation prior to 8% PEG6000 enrichment. The extracted exosomes were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting. Results The pellets obtained were round or elliptic membrane vesicles 30 to 150 nm in diameter with intact double membranes and contained low electron density material. The pellets expressed CD63, ALIX and TSG101 molecules but not calnexin that was expressed in sperm cells. Conclusion The PEG6000-based method for extraction of exosomes from semen samples facilitates future studies of seminal exosomes.