A critical stage of sperm maturation, termed ‘capacitation’, occurs while spermatozoa transit the female reproductive tract, and involves a drastic remodelling of the sperm surface architecture that ultimately affords spermatozoa the ability to recognise and fertilise an egg. Intact DNA and a lack of oxidative stress damage are hallmark characteristics of spermatozoa that successfully complete this process. These cells retain the ability to organise surface receptors that permit the specialised process of binding to the zona pellucida, just prior to fertilisation. This natural gamete selection process is bypassed in higher technology assisted reproduction and remains an untapped basis for improving sperm selection in the clinic. Despite decades of research, the entities responsible for sperm-egg binding are yet to be fully elucidated, which has greatly limited the translation of this platform. Thus, we have utilised comparative proteomic profiling, in silico analysis and immuno-staining to identify membrane proteins involved in mouse sperm surface remodelling. Mouse spermatozoa were isolated both immediately from the epididymis (non-capacitated) and after in vitro capacitation (capacitated). For proteomic analysis, membranes were isolated and comparatively characterised via high-resolution LC-MS/MS using label-free quantification and in silico subcellular stratification. We identified 884 and 787 membrane proteins from non-capacitated and capacitated spermatozoa, respectively. In addition to well-characterised membrane proteins such as molecular chaperones, we identified 207 proteins not previously described or annotated in the sperm membrane. Of the proteins identified, 146 and 49 were localised uniquely to the cell membrane in non-capacitated and capacitated spermatozoa, respectively. Subsequent validation of membrane proteins feature the carbohydrate binding protein, malectin, an egg recognition candidate with affinity for the sugars adorning the zona pellucida. This targeted characterisation of the sperm membrane during capacitation is providing a valuable resource for advancing our understanding of sperm maturation and the rational development of new sperm selection materials for ART.