Background: Fortification of donor human milk (DHM) is required for optimal growth of very preterm infants, but there are concerns of more gut dysfunction and necrotizing enterocolitis (NEC) when using formula-based fortifiers (FFs), especially soon after birth. Intact bovine colostrum (BC) is rich in nutrients and bioactive factors, and protects against NEC in preterm pigs. We hypothesized that fortification of DHM with BC is superior to FFs to prevent gut dysfunction and infections when provided shortly after preterm birth. Methods: Two FF products, Enfamil (ENF; intact protein, vegetable oil) and PreNAN+Nutrilon (NAN; extensively hydrolyzed protein, maltodextrin), were compared with BC as fortifier to DHM fed to preterm pigs for 5 days. Results: Relative to the DHM+BC group, DHM+FF groups had higher diarrhea score and lower hexose uptake and lactase activity, and specifically the DHM+NAN group showed higher gut permeability, NEC score, more mucosa-adherent bacteria with altered gut microbiota structure (ie, lower diversity, increased Enterococcus, decreased Staphylococcus abundance). Both DHM+FF groups showed higher expression of intestinal cytokine and inflammation-related genes, more gut-derived bacteria in the bone marrow, lower density of mucin-containing goblet cells, and slightly higher colon lactate, stomach pH and acetate, and blood neutrophil-to-lymphocyte levels than the DHM+BC group. Conclusions: Used as a fortifier to DHM, BC is superior to FFs to support gut function, nutrient absorption, and bacterial defense mechanisms in preterm pigs. It is important to optimize the composition of nutrient fortifiers for preterm infants fed human milk.