To investigate the effects of dietary leucine supplementation on endoplasmic reticulum (ER) stress in the liver of early-weaning piglets, we explored the morphology of liver cells, expression pattern of ER stress-associated proteins, and effects on ER stress induced by early weaning under leucine treatment using transmission electron microscopy (TEM), nucleo-cytoplasmic fractionation and immunoblotting assays. The results showed that in the early weaning-induced ER stress model, the diameter of ER in the liver cells derived from 14-day-old early-weaning piglets was increased compared with the normal group, while the structure of ER was disordered, and the expression level of ER stress-associated protein XBP1 in the nucleus of liver cells was significantly up-regulated (P＜0.05). In the HepG2 cells cultured in vitro, the ER stress-associated proteins ATF4 and cleaved ATF6α were significantly up-regulated (P＜0.05) with the absence of either glucose or leucine in the medium, while after re-stimulation with leucine, ATF4 expression was further up-regulated in the nucleus of HepG2 (P＜0.05). By feeding weaning piglets with diets containing different levels of leucine, we found that dietary leucine levels had no significant effect on the expression of ATF4 and cleaved ATF6α in the nucleus of liver cells derived from 21-day-old non-weaning piglets (P＞0.05). After weaning 24 h, the expression of ATF4 in the nucleus of liver cells derived from piglets fed with diet containing 2.10% leucine was significantly higher than that of 1.66% leucine group (P＜0.05). In summary, early weaning can induce ER stress in the liver cells of piglets, and leucine plays crucial roles in regulating ER stress induced by early weaning.