Human milk oligosaccharides (HMO) are major components of breast milk that may have local effects in the gastrointestinal tract and systemic functions after being absorbed, both depending on their metabolism. Using preterm pigs, we investigated the metabolic fate of HMO in three experiments with two different HMO blends. In addition, we examined effects on the colonic microbiota in the presence or absence of necrotizing enterocolitis (NEC). Thus, preterm pigs (n = 112) were fed formula without or with HMO supplementation (5–10) g/L of a mixture of 4 (4-HMO) or >25 HMO (25-HMO) for 5 (Experiment 1 and 2) or 11 days (Experiment 3). Individual HMO were quantified in colon contents and urine using MALDI-TOF-MS (matrix-assisted laser desorption ionization mass spectrometry) and HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection). Microbial colonization was analyzed by sequencing of 16S rRNA gene tags. Intestinal permeability was measured by lactulose to mannitol ratio in urine. HMO supplemented to formula were detected in urine and colon contents in preterm piglets after 5 and 11 days in all three experiments. The amount of HMO excreted via the gut or the kidneys showed large individual variations. Microbial diversity in the colon changed from high levels of Firmicutes (dominated by Clostridium) at day 5 (Exp 2) to high levels of Proteobacteria dominated by Helicobacter and Campylobacter at day 11 (Exp 3). Colonic microbiota composition as well as HMO excretion pattern varied greatly among piglets. Interestingly, the 5-day supplementation of the complex 25-HMO blend led to low concentrations of 3-fucosyllactose (FL) and lacto-N-fucopentaose (LNFP) I in colonic contents, indicating a preferred utilization of these two HMO. Although the interpretation of the data from our piglet study is difficult due to the large individual variation, the presence of Bifidobacteria, although low in total numbers, was correlated with total HMO contents, and specifically with 2′FL levels in colonic content. However, early supplementation of formula with HMO did not affect NEC incidence.