This study investigated the effect of complexation with whey and casein protein, respectively, on post prandial absorption of vitamin D₃. For this purpose, Sprague-Dawley rats (n = 78) were administered 840 IU vitamin D₃ dissolved in ethanol and either (i) complexed with whey protein isolate (protein : vitamin ration 2 : 1), (ii) complexed with caseinate (protein : vitamin ration 2 : 1), or (iii) provided in a water solution. Serum concentrations of vitamin D₃, 25-hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃ were measured before and 2, 3, 4, 5, 6, 7, 8, and 10 hours after administration of vitamin D₃. Significant effects of complexation on serum concentrations of vitamin D₃, 25-hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃ were demonstrated. Complexation with whey protein isolate resulted in the fastest and highest absorption of vitamin D₃ while an effect of complexation with caseinate was evident but more modest and non-significant. In conclusion, the study demonstrates that complexation with milk proteins is an efficient strategy to enhance bioaccessibility of vitamin D₃.