Infant microbiota is influenced by numerous factors, such as delivery mode, environment, prematurity and diet (breast milk or formula). In addition to its nutritional value, breast milk contains bioactive substances that drive microbial colonisation and support immune system development, which are usually not present in infant formulas. Among these substances, polyamines have been described to be essential for intestinal and immune functions in newborns. However, their effect on the establishment of microbiota remains unclear. Therefore, the aim of the present study was to ascertain whether an infant formula supplemented with polyamines has an impact on microbial colonisation by modifying it to resemble that in breast-fed neonatal BALB/c mice. In a 4 d intervention, a total of sixty pups (14 d old) were randomly assigned to the following groups: (1) breast-fed group; (2) non-enriched infant formula-fed group; (3) three different groups fed an infant formula enriched with increasing concentrations of polyamines (mixture of putrescine, spermidine and spermine), following the proportions found in human milk. Microbial composition in the contents of the oral cavity, stomach and small and large intestines was analysed by quantitative PCR targeted at fourteen bacterial genera and species. Significantly different (P< 0·05) microbial colonisation patterns were observed in the entire gastrointestinal tract of the breast-fed and formula-fed mice. In addition, our findings demonstrate that supplementation of polyamines regulates the amounts of total bacteria, Akkermansia muciniphila, Lactobacillus, Bifidobacterium, Bacteroides–Prevotella and Clostridium groups to levels found in the breast-fed group. Such an effect requires further investigation in human infants, as supplementation of an infant formula with polyamines might contribute to healthy gastrointestinal tract development.