High motility spermatozoa are good for cryopreservation and artificial insemination (AI) of mammalian semen. In this study, normal motility (NM) and low motility (LM) Mediterranean buffalo spermatozoa were compared using quantitative proteomics and phosphoproteomics techniques to screen for important proteins and phosphorylated proteins related to the motility of spermatozoa and to identify candidate protein molecular markers related to the quality of Mediterranean buffalo semen. Proteomics results identified 2550 proteins, with 119 proteins upregulated and 146 proteins downregulated in the LM spermatozoa versus the NM spermatozoa. The differentially abundant proteins were mainly involved in carbohydrate metabolism, glycolysis/gluconeogenesis, and tricarboxylic acid cycles. The phosphoproteomics analysis revealed 412 proteins, 1228 phosphorylated peptides, and 1465 phosphorylation modification sites. Compared to the NM group, 119 peptides were downregulated in the LM group, corresponding to 98 proteins, and 84 phosphorylated peptides were upregulated in the white matter, corresponding to 61 proteins. Differentially phosphorylated proteins were primarily involved in spermatogenesis, flagellate sperm motility, and glycolysis/gluconeogenesis. The combined proteomics and phosphoproteomics results identified the common proteins HMGB4, POC1B, PKM, LDHA, TBC1D21, and CBY2, whose main roles were related to spermatogenesis, sperm flagellar structure, and energy metabolism, which can be used as potential markers of Mediterranean buffalo sperm quality.