Objective To explore a new method for isolating proteins released by spermatozoa of pig epididymis, and determine the functional characteristics and signaling pathways of released proteins.Method The unique co-culture system of Transwell chamber was used to separate proteins secreted by sperms through 0.4 μm polycarbonate membrane. The isotopically labeled relative and absolute quantitation (iTRAQ) technique was used to analyze the released proteins and identify the differential proteins.The most important biochemical metabolic pathway and signal transduction pathway of differential proteins were confirmed by GO database describing their functions and KEGG database analyzing their pathways. Meanwhile, IPR database was used to conduct non-redundancy analysis for the domains of differential proteins.Result A total of 542 proteins were identified in the upper and lower rooms of Transwell chamber, of which 464 proteins were significantly up-regulated and 78 proteins were significantly down-regulated. The differential proteins were mainly involved in biological processes such as transmembrane transport, ion transport and ATP synthesis. The main involved signaling pathways of differential proteins were aldosterone-mediated sodium salt uptake, vesicle transport, and EGFR tyrosine kinase inhibition. Sulfur-oxidized protein, galactose, glycoside hydrolase and other major domains that constituted differential proteins provided structural bases for protein functions.Conclusion The method of separating proteins using Transwell chamber is effective. iTRAQ technique successfully identifies the differential proteins in proteins released by spermatozoa in epididymal head. The difference analysis and function annotation lay the foundation for exploring life activities and functional roles that differential proteins involved.