In this study, we conducted a comprehensive quantitative phosphoproteomic analysis of mouse epididymal sperm from different regions (caput, corpus, and cauda) to unveil the dynamics of protein phosphorylation during sperm maturation. And we performed phosphoproteomic analysis of ALK inhibitor 2 compound 18-treated and control sperm to gain further insights into the potential mechanisms by which TSSK2 regulates sperm motility.