We tested the effect of vagus nerve stimulation in endotoxin-induced intestinal tight junction injury in mice challenged with lipopolysaccharide (LPS) and examined the role of α7 nicotinic acetylcholine receptors (α7nAchR) in this process. Endotoxemia was induced by intraperitoneal injection of LPS (10 mg/kg) in male Balb/c mice. Samples were collected 12 h after LPS treatment. Endotoxemia was associated with intestinal barrier dysfunction, as evidenced by increased amount of fluorescein isothiocyanate-dextran in circulation. Western blot and immunofluorescence was performed, and the results demonstrated decreased expression of occludin and zonula occludens 1 along intestinal epithelium in endotoxemic mice. The ultrastructure of tight junction was disrupted as shown by transmission electron microscopy, which was associated with increased intestinal permeability. Stimulation of the right cervical vagus nerve ameliorated the damage of tight junction ultrastructure, which was consistent with decreased permeability to fluorescein isothiocyanate-dextran, and also reversed the decreased expression of tight junction proteins occludin and zonula occludens 1. Vagus nerve stimulation inhibited the upregulated activity of myosin light chain kinase and nuclear factor κB. In contrast, α-bungarotoxin (a specific α7nAchR antagonist, 0.1 μg/mouse) administered before vagus nerve stimulation significantly abolished these protective effects of vagus nerve stimulation. Our results for the first time confirmed that vagus nerve stimulation attenuated the disruption of tight junction in intestinal epithelium in endotoxemic mice, which was mediated through suppressing translocation of nuclear factor κB p65, downregulating myosin light chain kinase, and the α7nAchR may play an important role in this process.