Spermatozoa are functionally inert when they emerge from the testes. Functional competence is conferred upon these cells during a post-testicular phase of sperm maturation in the epididymis. Remarkably, this functional transformation of epididymal spermatozoa occurs in the absence of nuclear gene transcription or protein translation. To understand the cellular mechanisms underpinning epididymal maturation, we have performed a label-free, MS-based, comparative quantification of peptides from caput, corpus and caudal epididymal spermatozoa. In total, 68 phosphopeptide changes could be detected during epididymal maturation corresponding to the identification of 22 modified proteins. Included in this list are the sodium-bicarbonate cotransporter, the sperm specific serine kinase 1, AKAP4 and protein kinase A regulatory subunit. Furthermore, four phosphopeptide changes came from Izumo1, the sperm-egg fusion protein, in the cytoplasmic segment of the protein. 2D-PAGE confirmed that Izumo1 is post-translationally modified during epididymal transit. Interestingly, phosphorylation on Izumo1 was detected on residue S339 in the caput and corpus but not caudal cells. Furthermore, Izumo1 exhibited four phosphorylated residues when spermatozoa reached the cauda, which were absent from caput cells. A model is advanced suggesting that these phospho-regulations are likely to act as a scaffold for the association of adaptor proteins with Izumo1 as these cells prepare for fertilization.