The gut microbiota plays an important role in the postnatal development of the intestinal epithelium. Proteobacteria and Fusobacteria are abundant in the gut microbiota of neonatal piglets. We hypothesized that members of these phyla could regulate epithelial homeostasis in early life. Piglets received every day from birth to day 7 an oral administration of water (negative control, n=18 piglets from 3 litters) or colistin (antibiotic targeting specific gram negative bacteria, n=18 piglets from 3 litters). At day 7, we analysed the microbiota and the metabolome in the colon and we isolated epithelial cells for gene expression profiling and organoid culture. Microbiota richness and β-diversity were analysed with the R package ‘Phyloseq’. Linear mixed models were used to compare groups by including the litter as a random effect. Differential abundance analysis showed that colistin reduced the relative abundance of Proteobacteria (Enterobacteriaceae, Pasteurellaceae) and Fusobacteria (Fusobacteriaceae) while it did not change the microbiota richness. The modification of the microbiota composition induced by colistin was associated with a modification of the colon metabolome. The treatment with colistin increased the relative concentration of succinate, malonate and serotonin while it reduced the relative concentration of histamine, propionate and numerous lipids. Piglets treated with colistin expressed lower levels of genes involved in innate immunity such as Toll-like receptor 4 (TLR4) and lysozyme (LYZ) in the colon epithelium. This gene expression pattern persisted in piglet colon organoids after two passages, indicating that the depletion of Proteobacteria and Fusobacteria in vivo imprinted the epithelial stem cells from which organoids were derived. In conclusion, an early life modification of the microbiota altered the metabolome, epithelial gene expression and imprinted intestinal stem cells in the colon of piglets. Additional studies will be required to unravel the underlying mechanisms and to evaluate the long-term consequences for gut health in piglets.