Intestinal ischemia-reperfusion (I/R) injury can occur in clinical settings such as organ transplantation, cardiopulmonary bypass and trauma. The noble gas helium attenuates I/R injury in a number of animal organs and thus may offer a strategy for reducing I/R-induced intestinal injury in clinical settings. In the present study, we used four different helium preconditioning (HPC) profiles to investigate the potential beneficial effect of HPC on I/R-induced intestinal injury. Male Sprague-Dawley rats were pretreated with three cycles of air breathing for 5 min combined with three cycles of breathing a 70% helium:30% oxygen mixture for either 2, 5, 10, or 15 min, after which they were subjected to 60-min intestinal ischemia and 60-min reperfusion. Sixty minutes after reperfusion, the intestinal tissues of the variously treated rats were analyzed using histology, immunohistochemistry, terminal dUTP nick-end labeling staining, myeloperoxidase activity assay, Western blotting, and enzyme-linked immunosorbent assay for tumor necrosis factor α and macrophage inflammatory protein 1α. Intestinal permeability was assayed by measuring fluorescein isothiocyanate-dextran release in blood samples. The results showed that the HPC profile consisting of three cycles of 10 or 15 min of helium breathing and three cycles of 5 min of air breathing reduced I/R-induced intestinal injury, cell apoptosis, and the inflammatory response. However, the 2- or 5-min helium breathing did not confer any protective effects. It seems that longer helium episodes should be used in HPC profiles designed to attenuate intestinal I/R injury.