Subcellular proteomics not only deepens our knowledge of what proteins are present within cells, but also opens our understanding as to where those proteins reside. Given the highly differentiated, cross-linked state of spermatozoa, such studies have proven difficult to perform. In this study we have fractionated spermatozoa into two components, consisting of either the head or flagellar region. Following SDS-PAGE, 1 mm slices were digested and used for LC-MS/MS analysis. In total, 1429 proteins were identified with 721 proteins being exclusively found in the tail and 521 exclusively in the head. Not only is this the largest reported proteomic analysis of human spermatozoa, but also it has provided novel insights into the compartmentalization of proteins, particularly receptors, never previously reported to be present in this cell type.