The mammalian epididymis is an important male accessory gland where the spermatozoa gain the ability to fertilise the egg. To further understand the effects of testicular factors on the epididymis, the proteome of castrated adult mice and sham controls was compared using high-resolution two-dimensional gel electrophoresis following identification of proteins by matrix-assisted laser desorption ionisation time-of-flight/time-of-flight mass spectrometry. Twenty-three differentially expressed proteins (11 upregulated and 12 downregulated) were identified in epididymides from castrated. Bioinformatic analysis indicated that these castration-responsive proteins participated in energy metabolism and the antigen processing and presentation pathway. The differential expression levels were further validated by western blotting. The differentially expressed proteins may serve as potential candidates in studies of epididymal function and male infertility.