BACKGROUND: Human seminal fluid contains small exosome-like vesicles called prostasomes. Prostasomes have been reported previously to play an important role in the process of fertilization by boosting survivability and motility of spermatozoa, in addition to modulating acrosomal reactivity. Prostasomes have also been reported to present with sizes varying from 50 to 500 nm and to have multilayered lipid membranes; however, the fine morphology of prostasomes has never been studied in detail. METHODS: Sucrose gradient-purified prostasomes were visualized by cryo-electron microscopy (EM). Protein composition was studied by trypsin in-gel digestion and liquid chromatography/mass spectrometry. RESULTS: Here we report for the first time the detailed structure of seminal prostasomes by cryo-EM. There are at least three distinct dominant structural types of vesicles present. In parallel with the structural analysis, we have carried out a detailed proteomic analysis of prostasomes, which led to the identification of 440 proteins. This is nearly triple the number of proteins identified to date for these unique particles and a number of the proteins identified previously were cross-validated in our study. CONCLUSION: From the data reported herein, we hypothesize that the structural heterogeneity of the exosome-like particles in human semen reflects their functional diversity. Our detailed proteomic analysis provided a list of candidate proteins for future structural and functional studies.