Human milk (HM) contains a wide range of components that make it the optimal source of nutrients for growing infants. However, many circumstances preclude the use of HM, leading to the use of ruminant milk-based infant formula (IF) to meet nutritional requirements. Maximizing the nutritional properties of IF would therefore provide important health benefits, as well as commercial opportunities in the dairy industry. It was recently shown that feeding rats either HM or IF differentially altered gene expression in caecal tissue, and affected the microbiome composition in the caecum and colon. In this follow-up study, the impact of HM and IF on the gut function of rats was further explored via metabolomics and metagenomics. Metabolomics analysis of caecal contents using accurate-mass, high-resolution mass spectrometry revealed 17 metabolic features that were differentially abundant between the two treatment groups, including some amino sugars (higher in HM-fed rats) and bile acids (higher in IF-fed rats). Using shotgun sequencing, metagenomic analysis of DNA found in the ileum, caecum and colon showed that several genera within the Proteobacteria phylum were relatively more abundant in rats fed HM, while other taxa that differed in relative abundance included Prevotella and Ruminococcus. Gene functions related to carbohydrate and amino acid metabolism were elevated in HM-fed rats, consistent with differences observed in the caecal-content metabolome. Overall, this study provides new insights into how HM and IF affect the intestinal microbiome and host, and contributes to the body of literature expected to ultimately enable the redesign of IF formulations to deliver more human milk-like benefits.