Updated project metadata. Tadpole-shaped sperm is formed from round spermatid by spermiogenesis, a process with dramatic morphological changes in the final stage of spermatogenesis in testis. Protein phosphorylation, as one of the most important post-translational modifications, can regulate spermiogenesis, however, there is a lack of systemic analysis for phosphorylation events in this process. By large-scale phosphoproteome profiling using IMAC and TiO2 enrichment, we identified 18,980 phosphorylation sites in 4,628 phosphoproteins in mouse purified spermatids undergoing spermiogenesis. The identified phosphoproteins were significantly enriched in RNA transport, cell-cell adhesion, centrosome, microtubule and cilliogenesis. Further characterization of the kinase substrate relationship network demonstrated enrichment of phosphorylation substrates were related to the regulation of spermiogenesis. This global protein phosphorylation landscape of spermiogenesis showed wide phosphoregulation of diverse processes during spermiogenesis and could help further characterization of the process of sperm generation.