Objective To explore the activation effect of casein (CN) on neutrophils and its interaction with TLR4 receptors on the neutrophil surface.Methods The zebrafish model is adopted to study the activation effect of CN on neutrophil migration, with such methods as 3D model construction, molecular docking, and molecular dynamics simulations employed to study the interaction between TLR4 receptors and CN subtypes.Results Treatment of 3 dpf zebrafish with 1 mg/mL of CN solution for 2 h can induce neutrophil migration and accumulation inside the body of the zebrafish. All subtypes of CN can form stable complexes with TLR4. It is speculated that the key amino acids that maintain the stability of the complexes between α_S1-CN/α_S2-CN and TLR4 mainly bind via hydrogen bonds and hydrophobic interactions, while the key amino acids that maintain the stability of the complexes between β-CN/κ-CN and TLR4 mainly bind by hydrogen bonds.Conclusion CN can bind to TLR4 via hydrogen bonds and hydrophobic interactions, and mediate the activation of neutrophils, thus exerting its immunomodulatory effects.