Sepsis is the most common morbidity in preterm infants, who often receive total parenteral nutrition (TPN). _. We hypothesized that gut barrier function is compromised in TPNfed compared to enterally fed newborn piglets. Colostrum-deprived, newborn pigs were implanted with jugular venous and bladder catheters under general anesthesia. Pigs were either administered TPN (n=15) or fed formula (ENT, n=15). After 6 d, pigs were gavaged a solution of mannitol, lactulose, and polyethylene glycol-4000 (PEG 4000) and urine was collected for 24 h. At 7 d, small bowel samples were assayed for myeloperoxidase (MPO) activity, morphometry, and tight junction (TJ) protein abundance. Intestinal contents and peripheral organ sites were cultured for bacteria. Urinary recovery (%dose) of mannitol (53 vs. 68) was lower, whereas that of lactulose (2.93 vs. 0.18) and PEG4000 (12.78 vs. 0.96) were higher in TPN versus ENT pigs (p<0.05). The incidence of translocation was similar in TPN and ENT pigs. MPO activity was increased in TPN versus ENT pigs in the jejunum (p<0.001) and was weakly correlated with lactulose (R²=0.32) and PEG 4000 (R²=0.38) recovery. Goblet cell (GC) counts did not change, but intraepithelial lymphocyte numbers decreased with TPN. Only claudin-1 protein abundance was increased in the TPN group. We conclude that TPN is associated with impairment of neonatal gut barrier function as measure by permeability, but not translocation.