One of the key processes that forms the basis of fertilisation is the tight interaction between sperm and egg. Both sperm and egg proteomes are known to evolve and diverge rapidly even between closely related species. Understanding the sperm proteome therefore provides key insights into the proteins that underpin the mechanisms involved during fertilisation and the fusion between sperm and egg, and how they can differ across individuals of the same species. Despite being a commonly used model organism for reproductive research, little is currently understood about the zebrafish Danio rerio sperm proteome. We performed nanoLC-MS/MS proteomics analysis after off-line sample fractionation with six pooled samples containing sperm from ten males each. We confidently identified 5410 proteins, from which a total of 3900 GeneIDs were generated leading to 1720 Gene Ontology terms.