A very common conception about the function of the spermatozoon is that its unique role is to transmit the paternal genome to the next generation. Most of the sperm genome is known to be condensed in many species by protamines, which are small and extremely positively charged proteins (50–70% arginine) with the functions of streamlining the sperm cell and protecting its DNA. However, more recently, it has been shown in mammals that 2–10% of its mature sperm chromatin is also associated to a complex population of histones and chromatin-associated proteins differentially distributed in the genome. These proteins are transferred to the oocyte upon fertilization and may be involved in the epigenetic marking of the paternal genome. However, little information is so far available on the additional potential sperm chromatin proteins present in other protamine-containing non-mammalian vertebrates detected through high-throughput mass spectrometry. Thus, we started the present work with the goal of characterizing the mature sperm proteome of the European sea bass, with a particular focus on the sperm chromatin, chosen as a representative of non-mammalian vertebrate protamine-containing species. Proteins were isolated by acidic extraction from purified sperm cells and from purified sperm nuclei, digested with trypsin, and subsequently the peptides were separated using liquid chromatography and identified through tandem mass spectrometry. A total of 296 proteins were identified. Of interest, the presence of 94 histones and other chromatin-associated proteins was detected, in addition to the protamines. These results provide phylogenetically strategic information, indicating that the coexistence of histones, additional chromatin proteins, and protamines in sperm is not exclusive of mammals, but is also present in other protamine-containing vertebrates. Thus, it indicates that the epigenetic marking of the sperm chromatin, first demonstrated in mammals, could be more fundamental and conserved than previously thought. Abbreviations: AU-PAGE: acetic acid–urea polyacrylamide gel electrophoresis; CPC: chromosomal passenger complex; DTT: dithiothreitol; EGA: embryonic genome activation; FDR: false discovery rate; GO: Gene Ontology; IAA: iodoacetamide; LC: liquid chromatography; LC-MS/MS: liquid chromatography coupled to tandem mass spectrometry; MS: mass spectrometry; MS/MS: tandem mass spectrometry; MW: molecular weight; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate buffered saline; SDS: sodium dodecyl sulfate; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCA: trichloroacetic acid.