ABSTRACT: ?-Conglycinin (?-CG), an anti-nutritional factor, is a major allergen in soybeans to induce intestinal dysfunction and diarrhea in neonatal animals, including piglets and human infants. This study with a piglet model determined the effects of N-acetylcysteine (NAC) on intestinal function and autophagy in response to ?-CG challenge. Twenty-four 12-day-old piglets (3.44?±?0.28 kg), which had been weaned at 7 days of age and adapted for 5 days after weaning, were randomly allocated to the control, ?-CG, and ?-CG?+?NAC groups. Piglets in the control group were fed a liquid diet containing 10% casein, whereas those in the ?-CG and ?-CG?+?NAC groups were fed the basal liquid diets containing 9.5% casein and 0.5% ?-CG for 2 days. Thereafter, pigs in the ?-CG?+?NAC group were orally administrated with 50 mg (kg BW)-1 NAC for 3 days, while pigs in the other two groups were orally administrated with the same volume of sterile saline. NAC numerically reduced diarrhea incidence (-?46.2%) and the concentrations of hydrogen peroxide and malondialdehyde, but increased claudin-1 and intestinal fatty-acid binding protein (iFABP) protein abundances and activities of catalase and glutathione peroxidase in the jejunum of ?-CG-challenged piglets. Although ?-CG challenge decreased the villus height, villus height/crypt depth ratio, and mRNA levels of claudin-1 and occludin, no significant differences were observed in these indices between the control and ?-CG?+?NAC groups, suggesting the positive effects of NAC supplementation on intestinal mucosal barrier function. Moreover, NAC increased the concentrations of citrulline and D-xylose in the plasma, as well as the expression of genes for aquaporin (AQP) 3, AQP4, peptide transporter 1 (PepT1), sodium/glucose co-transporter-1 (SGLT-1), potassium inwardly-rectifying channel, subfamily J, member 13 (KCNJ13), and solute carrier family 1 member 1 (SLC1A1) in the jejunum, demonstrating that NAC augmented intestinal metabolic activity and absorptive function. Remarkably, NAC decreased Atg5 protein abundance and the LC3II/LC3I ratio (an indicator of autophagy) in the jejunum of ?-CG-challenged piglets. Taken together, NAC supplementation improved intestinal function and attenuated intestinal autophagy in ?-CG-challenged piglets.