Infant formulas (IFs), the only adequate substitute to human milk, are complex matrices that require numerous ingredients and processing steps. Previously, we showed that the quality of the dairy protein ingredients within IFs modulated protein microstructure and in vitro and in vivo digestive kinetics. Therefore, the aim was to assess the impact of protein ingredient quality (composition and structure) on gut physiology and microbiota. Three isonitrogenous IFs were formulated with whey proteins from different origins (cheese whey: IF-A, vs. ideal whey: IFs-B/C) and casein with different organizations (micellar: IFs-A/B, vs. non-micellar: IF-C). Twenty-four Yucatan mini-piglets (2- to 21-day-old), used as an infant model, received one of the three IFs. Digestive contents, faeces, and tissues were analysed using metagenomic, histological, ex vivo permeability and gene expression approaches and a metabolomic analysis was done on serum. Univariate and multivariate statistical analyses were performed. The increase of colonic paracellular permeability associated with a slight immune boost, and the changes in colonic amino acid transporter expression in IF-C compared with IF-A fed piglets, suggested a combined effect of whey origin and casein supramolecular organization on intestinal physiology in favour of IF-C, whose parameters were closer to those recently reported for human milk-fed piglets (Charton et al., 2022). Gut microbiota composition was moderately changed between diets, whereas faecal short-chain fatty acid composition differed according to whey protein origin. Differences in microbiota fermentative activity may result from differences in digestive kinetics previously observed in vitro between cheese whey-based IF (A) and ideal whey-based IFs (B and C), which could modulate the colonic substrate available for the microbiota. Serum metabolomic data are currently being analysed and may provide a better understanding of the mechanisms involved. The use of ideal whey and the modulation of casein organization appear to be possible avenues for improving IFs.