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Seminal plasma contains many morphologically heterogeneous extracellular vesicles (sEVs). These are sequentially released by cells of the testis, epididymis and accessory sex glands, and involved in male and female reproductive processes. This study aimed to in-depth define sEV-subsets isolated by ultrafiltration and size-exclusion chromatography (SEC), decode their proteomic profiles using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to quantify identified proteins using Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS). The sEV-subsets defined Large (L-EVs) or Small (S-EVs) by their protein concentration, morphology, size distribution and EV-specific protein markers and purity. LC-MS/MS identified a total of 988 proteins, 737 of them quantified by SWATH in S-EVs, L-EVs and non-EVs-enriched samples (18-20 SEC-eluted fractions). The differential expression analysis revealed 197 differentially abundant proteins between both EV-subsets, S-EVs and L-EVs, and 37 and 199 between S-EVs and L-EVs vs non-EVs-enriched samples, respectively. The gene ontology (GO) enrichment analysis of differentially abundant proteins suggested, based on the type of protein detected, that S-EVs could be mainly released through an apocrine blebbing pathway and be involved in modulating the immune environment of the female reproductive tract as well as during sperm-oocyte interaction. In contrast, L-EVs could be released by fusion of multivesicular bodies with the plasma membrane becoming involved in sperm physiological processes, such as capacitation and avoidance of oxidative stress. In conclusion, this study provides a procedure capable of isolating of subsets of EVs from pig seminal plasma with a high degree of purity and shows differences in the proteomic profile between EV-subsets,indicating different sources and biological functions for the sEVs.