BACKGROUND: Preterm (PT) infants harbor a different gut microbiome than full-term infants. Multiple factors affect gut microbial colonization of PT infants, including low gestational age, high rates of Cesarean section, exposure to antibiotics, and diet. Human milk, whether it's mother's own milk (MOM) or donor human milk, is the preferred feeding mode for PT infants but needs to be fortified to achieve adequate nutrient content. Infant formulas are introduced at later stages if human milk is insufficient or unavailable. How these dietary exposures affect the gut microbiome of PT infants is poorly understood. OBJECTIVES: The goal of this study was to evaluate the metagenomic potential of the fecal microbiome of PT infants consuming MOM with bovine milk-based fortifier compared with PT formula alone. METHODS: Forty-two stool samples, from 27 infants consuming MOM or formula (21 samples in each group) were included. Twelve infants had repeated sampling (2-3 samples). Shotgun genomic DNA sequencing was performed and analyzed using MetaPhlAn and HUMAnN2. Multivariate regression analysis, adjusting by the repeated sampling, was used to identify the features that differed between PT infants consuming MOM compared with formula. RESULTS: The primary function of the fecal microbiome of PT infants was characterized by a high abundance of biosynthesis pathways. A set of core features was identified; these belonged to pathways for amino acid metabolism and vitamin K-2 biosynthesis. Five pathways significantly differed between the MOM and formula group. Pathways for fatty acid and carbohydrate degradation were significantly higher in the MOM group. Taxonomically, members of the phylum Actinobacteria and the genus Bifidobacterium were higher in PT infants exposed to MOM. CONCLUSIONS: This study provides insight into the influence of feeding MOM compared with infant formula on the structure and function of the fecal microbiome of PT infants.