N-acetyl cysteine (NAC) has been widely used for preventing reactive oxygen species-induced damage. However, little is known as to whether dietary NAC supplementation would alleviate intestinal injury in weaned piglets. The present study evaluated the effect of NAC on enterocyte apoptosis and intracellular signalling pathways' response to weaning stress. The control piglets were normally suckling, and piglets in the weaning and NAC groups were fed the basal diet and basal+NAC diet from 14 to 25 d of age, respectively. Compared with the control piglets, weaning increased cortisol concentrations (P< 0·05), decreased superoxide dismutase and glutathione peroxidase activities (P< 0·05), increased malondialdehyde content (P< 0·05) in serum and enhanced enterocyte apoptosis index (AI) and concentrations of caspase-3, caspase-8 and caspase-9 (P< 0·05). Gene expression analyses indicated that weaning induced apoptosis via Fas signalling and mitochondrial pathways in weaned piglets. Dietary NAC supplementation decreased (P< 0·05) cortisol concentrations and the AI, increased (P< 0·05) antioxidant status in serum and alleviated histopathological changes in the intestine. It also inhibited Fas, caspase-3, caspase-8 and integrin αvβ6 (αvβ6) gene expressions in the NAC-treated piglets. However, no significant decrease (P>0·10) in caspase-3, caspase-8 and caspase-9 concentrations was observed in the NAC group compared with the weaning group. In conclusion, weaning may induce enterocyte apoptosis via the activation of Fas-dependent and mitochondria-dependent apoptosis. Although NAC had no effect on caspase concentrations, it was clearly beneficial for preserving morphological integrity in weaned piglets via the regulation of cell apoptosis and the inhibition of Fas-dependent apoptosis and αvβ6 expression.