Sperm develop within the seminiferous tubules of the testis. Outside the tubules, the interstitium contains Leydig and immune cells, and blood and lymphatic vessels. Sperm first appear at puberty after the development of immune tolerance and must be protected from the immune system to prevent recognition as “non-self”. A major protective component is the blood-testis barrier (BTB), comprised of tight junctions between Sertoli cells which prevent free passage of proteins and create a specialised milieu for sperm development. Sperm proteins in healthy men are widely assumed to be sequestered inside the BTB, and physically prevented from interacting with the immune system or entering the circulation. To address this unproven assumption, we performed the first in-depth proteomic analysis of testicular interstitial fluid (TIF) in mice and humans, and defined the impact of the seminiferous epithelium on the TIF proteome. We show that, in both mice and men, TIF contains hundreds of proteins predominantly or specifically expressed by developing sperm inside the BTB, and many of these are reduced in mouse TIF during seminiferous epithelial disruption. In silico analysis revealed some sperm-specific TIF proteins are detectable in human plasma, contradicting the dogma that these proteins are absent from the circulation. TIF also contains numerous cancer-testis antigens (CTAs); sperm-specific proteins aberrantly expressed in cancers. CTAs are thought to be neoantigens and absent from the circulation, hence are considered ideal targets for cancer diagnosis and/or immunotherapy. However our data suggests certain CTAs are present in the circulation and in contact with the immune system. In conclusion, we reveal a conserved feature of the testis is the deposition of sperm proteins into TIF that can then enter the circulation. These findings have major implications for the development of CTA-based diagnostics and therapeutics, and provide hitherto unrecognised opportunities for new diagnostics to monitor testis function.