This study employed proteomics and phosphoproteomics to compare protein expression and phosphorylation modifications in the milk fat globule membrane (MFGM) of human and bovine mature milk. A total of 410 proteins and 73 phosphorylation sites on 49 proteins were identified in the MFGM of bovine and human mature milk, respectively. Differential analysis revealed 40 differentially expressed proteins and 8 differentially phosphorylated sites, followed by bioinformatics analysis. Seven overlapping proteins were identified, with GLYCAM1 and Alpha-S2-casein showing significant differences in both protein expression and phosphorylation modifications, particularly in relation to immune functions. The results suggest that bovine MFGM may provide unique advantages in supporting infant immune systems and promoting gut health, offering valuable insights for future innovation in infant formula development.