Technical Abstract: Total parenteral nutrition (TPN) has been associated with sepsis, translocation of gut luminal bacteria, gut mucosal atrophy, impaired gut barrier function, and increased gut permeability in preterm human infants. In this study, we sought to compare the composition of microbiota in the neonatal pig gut to translocation events and to examine the effects of TPN versus enteral (ENT) feeding on early gut colonization. Newborn, colostrum-deprived pigs (<24 h old) were fitted with intravenous catheters and divided into two groups. One group (n = 13) received total TPN through intravenous feeding and the second group (n = 13) (enteral-ENT) was fed a commercial milk replacer. After 7 d of treatment, pigs were euthanized and samples were collected for bacterial culture from the blood, intestinal tract and organs. ENT pigs had increased bacterial diversity (number of species isolated), higher concentrations of bacteria, and bacterial colonization of all segments of the intestinal tract compared to the TPN pigs. Translocation of bacteria from the intestinal tract to tissues or blood was similar for both groups. The ENT group had 1/13 positive for Clostrium difficile toxin A whereas the TPN group had 5/13. We concluded that TPN was not positively associated with translocation, that ENT favored increased bacterial concentrations comprised of more speciation in the gastrointestinal tract compared to TPN, and that TPN-treated neonates were at higher risk of colonization by toxin-expressing strains of C. difficile.