While advanced reproductive technologies (ARTs) are widely used in domestic animals, successful implementation of ARTs to conserve wildlife species remains challenging. In macropods, crucial aspects of fundamental reproductive biology, including changes induced by epididymal maturation, remain unknown, limiting the development of ARTs. In this context we performed a proteomic analysis of sperm from the caput, corpus, and cauda epididymis of Eastern Grey Kangaroos (n = 6) to profile changes during epididymal maturation. Samples prepared by FASP digestion were analysed by LC-MS/MS with SWATH acquisition. 3,889 proteins were identified overall. Proteins which were most abundant in sperm across all epididymal regions included ATP synthase F1 subunit alpha (ATP5F1A) and A-kinase anchoring protein (AKAP4). The top proteins in corpus and caput sperm were involved in protein binding, while the most abundant proteins in cauda sperm were involved in ATP metabolic processes and generation of precursor metabolites. Comparing corpus to caput sperm, 266 proteins differed significantly (104 increased, 162 decreased). Comparing corpus to cauda sperm, 502 proteins differed significantly (193 increased, 309 decreased). Proteins with differing abundance between regions largely had catalytic and binding activities. Proteins with higher abundance in caput and corpus sperm included lysosomal proteins (CTSC, NAAA) and chaperones (CALR, TXNDC5). From corpus to cauda, there was a clear increase in proteins involved with locomotion (CFAP47, RSPH6A, FER). Proteins involved in glycolysis (ENO1, GAPDH), mitochondrial ATP synthesis (ATP5F1A, CKB) and microtubule function (DNAH1, TEKT1) increased in abundance towards the cauda. These proteomic alterations likely underlie the significant sperm structural remodeling unique to marsupial epididymal transit (e.g. formation of midpiece fibre network, post-testicular acrosome formation). These results also support the necessity of completing epididymal maturation for development of full motility potential, including both energy production and cytoskeletal maturation.