Abstract: In order to investigate the effects of in vitro digestive properties and pro-calcium transport of three whole nutrient foods for special medical purposes with distinct protein compositions (non-hydrolyzed protein B₀, comprising approximately 55% whey protein and 45% casein, 100% non-hydrolyzed whey protein B₁, and 100% moderately hydrolyzed whey protein B₂). In vitro digestion study was conducted following the international INFOGEST method and the samples were subsequently analyzed for free α-amino acid content, degree of hydrolysis, peptide sequences, and amino acid composition. Furthermore, a Caco-2 monolayer cell model was established to explore the effects of three whole nutrient foods for special medical purposes on calcium transport both pre- and post-digestion. The results showed that when the digestion time was 240 min, the degrees of hydrolysis for non-hydrolyzed whey protein B₁ and moderately hydrolyzed whey protein B₂ were 50% and 45.5%, respectively, which were greater than that of non-hydrolyzed protein B₀ (the degrees of hydrolysis was 36.5%), and the average peptide chain lengths for B₁ and B₂ were 5.06 and 4.52, respectively, both shorter than that of B₀ (average peptide chain length of 5.45), suggesting that B₁ and B₂ exhibited greater in vitro digestibility. In addition, B₂ contained a larger number of amino acids that could bind calcium, and it demonstrated superior calcium transport ability prior to digestion, with a transport amount of 32.20 µg over a transit time of 120 min, while after digestion, B₁ exhibited enhanced calcium transport ability, with a transport amount of 35.70 µg at the same transit time. The bioavailability analysis of calcium revealed that the bioavailability of B₀ and B₂ before digestion, and B₁ after digestion were 32.76%, 36.39%, and 40.60%, respectively, all exceeding that of CaCl₂. These findings suggest that the three formulations above can effectively enhance calcium bioavailability and facilitate its absorption in the intestine. This study lays the groundwork for future investigations into whole nutrient foods for special medical purposes and provides valuable insights for the development of calcium supplement products.