Proteomics represents a powerful tool for the analysis of fish spermatozoa, since these cells are transcriptionally inactive. The aim of the present study was to generate an inventory of the most prominent rainbow trout sperm proteins with the use of one-dimensional electrophoresis prefractionation combined with performance liquid chromatography electrospray ionization tandem mass spectrometry. This study provides the first in-depth analysis of the rainbow trout sperm proteome, with a total of 204 identified proteins. We found that rainbow trout spermatozoa are equipped with functionally diverse proteins related to energetic metabolism, signal transduction, protein turnover, transport, cytoskeleton, oxidative injures and stress and reproduction. The availability of a catalogue of rainbow trout sperm proteins provides a crucial tool for the understanding of fundamental molecular processes in fish spermatozoa for ongoing research in the development of novel markers of sperm quality and for the optimization of short- and long-term sperm preservation procedures.