Spermatogenesis is a complex cell differentiation process that includes marked genetic, cellular functional and structural changes. It requires tight regulation, since disturbances in any of the spermatogenic stages would lead to fertility deficiencies. In order to increase our knowledge of signal transduction during sperm development, we carried out a large-scale identification of the phosphorylation events that occur in the human gonad. Metal oxide affinity chromatography using TiOx combined with LC-MS/MS was conducted to profile the phosphoproteome of human testes with full spermatogenesis. A total of 8187 phosphopeptides derived from 2661 proteins were identified, resulting in the most complete report of human testicular phosphoproteins to date. Phosphorylation events were enriched in proteins functionally related to spermatogenesis, as well as to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle and apoptosis. Moreover, 174 phosphorylated kinases were identified. The most active and abundant human protein kinases in the testis were predicted both by the phosphorylation status of the kinase activation loop and the number of phosphopeptide spectra identified. The potential function of two of those kinases, cyclin-dependent kinase 12 (CDK12) and p21-activated kinase 4(PAK4), has been explored by protein-protein interaction analysis, immunodetection in human and mouse testicular tissue, and functional assay in a human embryonal carcinoma cell line. The co-localization of CDK12 with Golgi markers and probably pro-acrosomal vesicles suggests a potential crucial role of this protein kinase in sperm formation. PAK4 expression has been found limited to human spermatogonia, and a role in embryonal carcinoma cell response to apoptosis has been observed. Together, our data confirm that phosphoregulation by protein kinases is highly active in sperm differentiation, and open a window to detailed characterization and validation of potential targets for the development of drugs modulating male fertility, and tumor behavior.