Arguably the most important tissue involved in post-testicular mammalian sperm maturation is the epididymis, a highly specialised ductal system that drives dramatic functional changes in spermatozoa. Indeed, it is during epididymal maturation that spermatozoa acquire the competence to engage in fertilisation and surprisingly, these changes occur in the complete absence of endogenous gene transcription or de novo protein translation. Despite years of study, we have yet to fully resolve the key pathways that facilitate and promote epididymal sperm maturation. To enhance our understanding of the factors that promote sperm maturation, we have sought to characterise the proteome of the caput, non-capacitated (NC) and capacitated (CAP) cauda epididymal spermatozoa. To achieve this we have employed a comparative and quantitative label-based proteomic approach coupled to high-resolution liquid chromatograph tandem mass spectrometry (LC-MS/MS). Preliminary analysis has achieved an improved depth of the sperm proteome, characterising 1,981 unique proteins (≥ 2 unique peptides). Strikingly, a total of 848 of these proteins were differentially expressed between the caput and the NC cauda spermatozoa and a further 144 had altered abundance between in the CAP vs NC spermatozoa (fold changes of ≤ -1.5 or ≥ 1.5; p ≤ 0.05). Alterations in the former comparison included ADAM3 (2.86 fold change) and threonine aspartase 1 (2.68 fold change) and in the later comparison, calcium and integrin-binding protein 1 (3.25 fold change) and glutathione S-transferase Mu 5 (2.65 fold change). Utilising Ingenuity Pathway Analysis, we assigned canonical pathways and upstream regulators to both comparisons, identifying rapamycin-insensitive companion of mTOR and transcription initiation factor TFIID subunit 7-like as activated upstream regulators with potential roles in sperm maturation. These preliminary analyses are providing us with the capacity to progressively track the proteomic changes associated sperm maturation and will help improve our understanding of human male fertility and ultimately infertility.