Early life nutrition fundamentally influences neonatal development and health. Human milk oligosaccharides (HMO) are key components of breast milk but not standard infant formula that support the establishment of the newborn gut microbiota. Using an artificial rearing system, our objective was to test the effect of two HMO on the whole body and organ growth, adiposity, glucose tolerance and faecal microbiota in young rat pups. From postnatal days 4 to 21, Sprague–Dawley rats were randomised to receive one of: (1) CTR (rat milk substitute); (2) 2′FL (CTR + 1·2 g/l 2′-fucosyllactose); (3) 3′SL (CTR + 1·2 g/l 3′-sialyllactose) and (4) 2′FL + 3′SL (CTR + 0·6 g/l 2′-FL + 0·6 g/l 3′-SL). Body weight (BW), bowel movements and food intake were monitored daily, faecal samples collected each week and oral glucose tolerance, body composition and organ weight measured at weaning. No significant differences were observed between groups in growth performance, body composition, organ weight and abundance of dominant faecal microbes. A decreased relative abundance of genus Proteus in week 1 faecal samples and Terrisporobacter in week 3 faecal samples (P < 0·05) was suggestive of a potential pathogen inhibitory effect of 3′SL. Longitudinal changes in the faecal microbiota of artificially reared suckling rats were primarily governed by age (P = 0·001) and not affected by the presence of 2′-FL and/or 3′-SL in rat milk substitutes (P = 0·479). Considering the known protective effects of HMO, further investigation of supplementation with these and other HMO in models of premature birth, extremely low BW or malnutrition may show more pronounced outcomes.