Milk has been used routinely as an extender for sperm preservation. Caseins, the major proteins in milk, are proposed to be the protective constituents of milk during sperm preservation. It is unclear whether the whey proteins in milk are also implicated in the protection of sperm. Our previous studies have shown that the major proteins of bovine seminal plasma (recently named as binder of sperm or BSP, which comprises BSP1, BSP3, and BSP5 proteins) mediate a continuous phospholipid and cholesterol efflux from the sperm plasma membrane that is detrimental for sperm preservation. In this study, we investigated whether the protective effect of milk could be due to an interaction between BSP proteins and milk proteins. The binding of BSP proteins to milk proteins was demonstrated by gel filtration chromatography. Milk was fractionated into three fractions: the first containing whey protein aggregates and kappa-casein, the second containing all milk proteins, and the third containing small peptides, salts, and sugars. BSP1 has a higher affinity for the milk proteins in the milk fractions as compared to BSP3 and BSP5. The binding of BSP proteins to milk proteins was further characterized by isothermal titration calorimetry. We demonstrated that BSP1 binds to caseins and the titration could be simulated with a Scatchard approach, leading to an affinity constant (K(a)) of 350 mM(-1) and a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin was characterized by a K(a) of 240 mM(-1) and an n value of 0.8. These results indicate the existence of an interaction between BSP proteins and milk proteins that could be the origin of the protection of sperm during preservation in milk.