Objective of this work was to study the effect of protein composition, and in particular, the presence of whey proteins (WP) or β-casein (β-cas), on the digestion behavior of a model infant formula. Three model formulas were prepared with the same concentration of protein, fat and lactose, as well as same caloric content. One formula contained only WP and 2 others contained a casein to WP ratio of 40:60, but differing by 24% in β-cas amount. The formula was obtained by mixing milk protein isolates and permeates prepared with a 80 KDa molecular weight cut-off membrane, and with addition of whey protein. An in vitro gastric digestion was performed to better understand the physicochemical properties of the gastric curd, and it was then followed by an intestinal digestion for studying physiological responses. There were no differences in the properties of the gastric digestate, within a treatment, at both 60 or 120 min of digestion. All curds showed a shear-thinning behavior, with a significantly higher viscosity and a higher modulus for curds obtained from casein/WP formula, compared with the curds from WP formula. Electrophoretic analysis of digestate samples after the gastric stage showed caseins extensively hydrolysed to peptides, while β-lg and α-la still largely intact after 60 or 120 min of digestion. Confocal microscopy showed structures with a higher density throughout the matrix for digestates obtained from cas/WP formula, compared with the WP formula digestate. The effect of in vitro intestinal digestate on secretion of pro-inflammatory cytokines was also studied on LPS stimulated human macrophages treated with the basolateral fraction of Caco-2 cells. Cas/WP formulas resulted in lower TNF-α secretion than WP formula. Moreover, high β-cas digestate showed lower secretion of IL-6, compared with WP and low β-cas formula. There was not a significant difference in secretion of IL-1β, between the 3 formulas. The results bring significant advances to our understanding of the effect of milk protein composition on the digestion of dairy matrices and physical properties of the gastric digestate, as well as their role on physiological responses.