Rat epididymal protein DE associates with the sperm surface during maturation and participates in sperm-egg fusion. Immunization of male rats with DE raised specific antibodies and produced a significant reduction in the animals' fertility. The present study focused on determining the in vivo mechanism involved in fertility inhibition. Wistar males were injected with DE, and antibody levels and animal fertility were evaluated. Results revealed an association between the two parameters, since animals with absorbance values lower than 0.5 in ELISA presented high fertility rates (66%, 100%) while those with absorbance values higher than 0.5 exhibited the lowest fertility rates (0%, 33%). Histological studies showed no evidence of orchitis, epididymitis, or vasitis in DE-immunized animals. ELISA results revealed the presence of anti-DE antibodies in epididymal and vas deferential fluids. Indirect immunofluorescence and ELISA experiments indicated that these antibodies would not interfere with the synthesis or secretion of DE or with its association with the sperm surface. Finally, while epididymal sperm recovered from DE-immunized animals presented no changes in motility, viability, or ability to undergo capacitation and acrosome reaction, they exhibited a significant decrease in their ability to fuse with zona-free eggs, with no effect on their ability to bind to the oolemma. Together these results indicate that immunization of male rats with epididymal protein DE specifically interferes with the sperm fertilizing ability, supporting the use of epididymal proteins for contraceptive vaccine development.