Early nutrition plays a dominant role in infant development and health. It is now understood that the infant diet impacts the gut microbiota and its relationship with gut function and brain development. However, its impact on the microbiota-gut-brain axis has not been studied in an integrative way. The objective here was to evaluate the effects of human milk (HM) or cow’s milk based infant formula (IF) on the relationships between gut microbiota and the collective host intestinal-brain axis. Eighteen 10-day-old Yucatan mini-piglets were fed with HM or IF. Intestinal and fecal microbiota composition, intestinal phenotypic parameters, and the expression of genes involved in several gut and brain functions were determined. Unidimensional analyses were performed, followed by multifactorial analyses to evaluate the relationships among all the variables across the microbiota-gut-brain axis. Compared to IF, HM decreased the α-diversity of colonic and fecal microbiota and modified their composition. Piglets fed HM had a significantly higher ileal and colonic paracellular permeability assessed by ex vivo analysis, a lower expression of genes encoding tight junction proteins, and a higher expression of genes encoding pro-inflammatory and anti-inflammatory immune activity. In addition, the expression of genes involved in endocrine function, tryptophan metabolism and nutrient transport was modified mostly in the colon. These diet-induced intestinal modifications were associated with changes in the brain tissue expression of genes encoding the blood-brain barrier, endocrine function and short chain fatty acid receptors, mostly in hypothalamic and striatal areas. The integrative approach underlined specific groups of bacteria (Veillonellaceae, Enterobacteriaceae, Lachnospiraceae, Rikenellaceae, and Prevotellaceae) associated with changes in the gut-brain axis. There is a clear influence of the infant diet, even over a short dietary intervention period, on establishment of the microbiota-gut-brain axis.