Protein phosphorylation, as one of the most important post-translational modifications, exerts crucial roles in regulating meiosis. However, there is a lack of systemic phosphoproteomic and functional analysis of phosphorylation in the meiosis of spermatocytes. To characterize the phosphorylation events in meiosis, we performed large-scale phosphoproteome profiling of purified spermatocytes undergoing meiosis, and identified 20,582 phosphorylation sites in 5,289 phosphoproteins, which were significantly enriched in cell cycle, autophagy, DNA repair, chromosome segregation. Kinase-substrate phosphorylation network analysis followed by in vitro meiosis study showed that CDK9 was a kinase with enriched substrate phosphorylation sites in spermatocytes, and was essential for meiosis progression to metaphase I. We also identified 15 new phosphorylation sites of histones, and 428 epigenetic factors, among which HASPIN was found to be essential for male fertility. Haspin knockout leaded to misalignment of chromosomes, apoptosis of metaphase spermatocytes and decreased number of sperm by deregulation of H3T3ph and Aurora-B kinase-containing chromosomal passenger complex. Our spermatocyte phosphoproteome will be a rich resource for future studies of phosphorylation signaling regulation of meiosis.