Colonization of the intestinal microbiota after birth plays an important role in development of the neonatal gastrointestinal and immune systems. Two key environmental factors that influence the colonization pattern are delivery mode and nutrition. In this study, the impact of delivery mode and nutrition on microbial colonization and metabolic activity was investigated in the pig model. Vaginally (VD) or caesarean- (CD) delivered piglets were sow-reared (SR) or fed formula alone (FF) or with 4 g/L prebiotics [1:1 ratio of short-chain fructo-oligosaccharides (scFOS) and polydextrose (PDX); FP]. Intestinal contents were collected on d 7 and 14. SR piglets harbored different microbial populations from FF and FP piglets in ileum and ascending colon (AC). On d 7, FF piglets had a greater abundance of Clostridium XIVa in AC, but lower total bacteria, Clostridium XIVa, and Lactobacillus spp. in ileum and Fecalibacterium prausnitzii in AC compared with FP piglets. On d 14, total bacteria were more abundant in FP than FF piglets. Butyrate, isobutyrate, valerate, and isovalerate concentrations in AC were greater in SR piglets compared with FF or FP piglets. At both sampling days, acetate concentrations in AC were similar between the SR and FF groups, whereas propionate was higher in the SR compared with FF group. Delivery mode also significantly affected microbial populations. Bacterial densities differed in AC for Bacteroides-Prevotella at d 7 and Clostridium XIVa at d 14, being higher in VD piglets. Correspondingly, VD piglets had higher propionate in ileum and propionate and butyrate in AC compared with CD piglets. Our results indicate that both delivery mode and nutrition affect microbial composition and metabolic activity. Supplementation of scFOS/PDX to formula modulates microbial colonization and produces a SCFA pattern closer to that of SR piglets.