β-Lactoglobulin (β-lg) is the main protein of ruminant milk whey. Although β-lg can bind in vitro to a variety of hydrophobic substrates, mainly retinol and long-chain fatty acids, its physiological function is still unknown. In Exp. 1, we investigated the effect of β-lg on the plasma retinol concentration in preruminant calves. Holstein male calves (n = 20) were fed Holstein whole milk at 40 g/kg body weight (BW) plus vitamin A acetate (500,000 i.u.) with or without β-lg (0·4 g/kg BW). The plasma retinol concentration of 10-d-old calves was greater (P<0·05) in the β-lg-fed group than in the control group during the period from 8 to 12 h and at 24 h after the feeding. The postprandial change of plasma retinol in 40-d-old calves fed milk with β-lg was higher (P<0·05) than that in the control calves only at 12 h after the feeding. In Exp. 2, Holstein male calves (n = 18) were used to investigate the effect of β-lg on plasma triglyceride concentration and fatty acid composition. Calves were fed Holstein whole milk at 40 g/kg BW plus milk fat prepared from whole milk at 2 g/kg BW with or without β-lg (0·4 g/kg BW). Plasma triglyceride concentration at age 10 d was higher (P<0·05) in the β-lg-fed group than in the controls during the periods from 1 to 2 h and from 7 to 11 h after the feeding. At age 40 d, plasma triglyceride in the β-lg-fed group was higher (P<0·05) than in the control group only at 9 h. Ratios of palmitic, stearic, and oleic acids to total plasma lipids were higher (P<0·05) in the calves fed β-lg milk than in the control calves at age 10 d. These results suggest that β-lg enhances the intestinal uptake of retinol, triglyceride, and long-chain fatty acids in preruminant calves.