Round haploid spermatids are formed at the completion of meiosis. These spermatids then undergo morphological and cytological changes during spermiogenesis. Although sperm proteomes have been extensively studied, relatively few studies have specifically investigated the proteome of round spermatids. We developed a label-free quantitative method in combination with 2D-nano-LC-ESI-MS/MS to investigate the proteome of round spermatids in mice. Analysis of the proteomic data identified 2,331 proteins in the round spermatids. Functional classification of the proteins based on Gene Ontology terms and enrichment analysis further revealed the following: 504 of the identified proteins are predicted to be involved in the generation of precursor metabolites and energy; 343 proteins in translation and protein targeting; 298 proteins in nucleotide and nucleic acid metabolism; 275 and 289 proteins in transport and cellular component organization, respectively. A number of the identified proteins were associated with cytoskeleton organization (183), protein degradation (116) and response to stimulus (115). KEGG pathway analysis identified 68 proteins that are annotated as components of the ribosomal pathway and 17 proteins were related to aminoacyl-tRNA biosynthesis. The round spermatids also contained 28 proteins involved in the proteasome pathway and 40 proteins in the lysosome pathway. A total of 60 proteins were annotated as parts of the spliceosome pathway, in which heterogeneous nuclear RNA is converted to mRNA. Approximately 94 proteins were identified as actin‑binding proteins, involved in the regulation of the actin cytoskeleton. In conclusion, using a label-free shotgun proteomic approach, we identified numerous proteins associated with spermiogenesis in round spermatids.