Understanding the cellular mechanisms that regulate mammalian sperm function is strategically important for both the management of male infertility and the development of novel approaches to male contraception. The spermatozoon is a transcriptionally and translationally suppressed cell that is released from the testes in a functionally inert state. Functional activation occurs in the epididymis and female tract via mechanisms that are entirely dependent on post-translational modifications. Proteomics is, therefore, the ideal technology to investigate this cell type. Herein, we comment on the proteomic analyses that have been applied to mammalian spermatozoa, including some concerns relating to data interpretation. Three comprehensive liquid chromatography-mass spectrometry lists of human, mouse and rat spermatozoa are then compared, insights into the molecular regulation of sperm function discussed and future directions speculated upon.