Background: β-casein is the main casein constituent in human milk (HM) and a source of bioactive peptides for the developing gastrointestinal tract and immune system. Infant formulas contain less β-casein than HM, but whether different concentrations of β-casein affect tolerability and gut and immune maturation in newborns is unknown. Objectives: Using near-term piglets as a model for newborn infants, we investigated whether increasing the β-casein fraction in bovine-based formula is clinically safe and may improve gut and immune maturation. Methods: Three groups of near-term pigs (96% gestation) were fed formula with bovine casein and whey protein (ratio 40:60): 1) standard skim milk casein (BCN-standard, 35% β-casein of total casein, n = 18); 2) β-casein enrichment to HM concentrations (BCN-medium, 65%, n = 19); and 3) high β-casein enrichment (BCN-high, 91%, n = 19). A reference group was fed 100% whey protein concentrate (WPC) as protein (WPC, n = 18). Intestinal and immune parameters were assessed before and after euthanasia on day 5. Results: Clinical variables (mortality, activity, body growth, and diarrhea) were similar among the groups, and no differences in intestinal or biochemical parameters were observed between BCN-standard and BCN-medium pigs. However, pigs receiving high amounts of β-casein (BCN-high) had lower small intestine weight and tended to have more intestinal complications (highest gut pathology score, permeability, and interleukin-8) than the other groups, particularly those receiving no casein (WPC pigs). Blood lymphocyte, thrombocyte, and reticulocyte counts were increased with higher β-casein, whereas eosinophil counts were reduced. In vitro blood immune cell responses were similar among groups. Conclusions: β-casein enrichment of bovine-based formula to HM concentrations is clinically safe, as judged from newborn, near-term pigs, whereas no additional benefits to gut maturation were observed. However, excessive β-casein supplementation, beyond concentrations in HM, may potentially induce gut inflammation together with increased blood cell populations relative to natural β-casein concentrations or pure whey-based formula.