The efferent ducts are a series of tubules that conduct sperm from the rete testis to the epididymis. They absorb most fluid and proteins originating from the rete testis during concentration of spermatozoa prior to their entry into the epididymis. Proteome analysis of micro-dissected efferent duct samples from adult rats was combined with genome-wide computational prediction of conserved hormone response elements to identify factors likely regulated by oestrogens and androgens. We identified 165 proteins and found subsets of the promoters controlling their corresponding genes to contain androgen- and oestrogen response elements (ARE/EREs) at similar frequencies. Moreover, EREs were significantly enriched among the loci identified compared with their genome-wide occurrence. The expression and localization of Anxa6, Ckb, Krt19, Park7, Pdzk1 and Tpt1 in the efferent ducts and other related hormone controlled tissues was further validated at the RNA or protein level. This study identifies many novel proteins predicted to play roles in sperm maturation and male fertility and provides significant computational evidence that the efferent ducts express genes transcriptionally controlled by sex hormones.