In this study, the relationship between milk clotting characteristics by rennet and different protein components was discussed. Addition of whey protein concentrate (WPC), κ-casein (κ-CN), β-lactoglobulin A (β-LgA) and β-lactoglobulin B (β-LgB) to skim milk could reduce the milk clotting time, increase the viscosity after coagulation and result in a more compact microstructure, while the formation of curds was inhibited by the addition of α-La, α-CN, β-CN and total CN and the microstructure of the formed curds was relatively loose. Dynamic light scattering method was applied to determine the particle size distribution of casein micelles during coagulation. The results showed that molecular radius distribution in casein solutions added with WPC, κ-CN, β-LgA and β-LgB changed quickly, indicating rapid aggregation of the protein molecules in solutions. Bidirectional electrophoresis and mass spectrometry identification of raw milk samples with significantly differential milk-clotting properties indicated that the differential protein components related to the rennet coagulation characteristics of bovine milk were mostly bioactive trace proteins affecting breast cell metabolism.