Oxidative stress (OS) plays a major role in the gastrointestinal disorders. Although probiotics were reported to repress OS, few researches compared the antioxidant ability of different Bacillus strains and deciphered the mechanisms. To select a Bacillus strain with higher antioxidant capacity, we used H2O2 to induce intestinal porcine epithelial cell 1 (IPEC-1) OS model. The most suitable H2O2 concentration and incubation time were determined by the half lethal dose and methyl thiazolyl tetrazolium. Correlation analysis was performed to choose a sensitive indicator for OS. As for the comparison of Bacillus, cells were divided into control, Bacillus treatment, H2O2 treatment, and Bacillus pre-protection + H2O2 treatment. Bacillus were co-cultured with IPEC-1 for 3 h in Bacillus and Bacillus pre-protection + H2O2 treatments. Then, based on OS model, 300 mu mol/L H2O2 was added into medium of H2O2 and Bacillus pre-protection + H2O2 treatments for another 12 h. Antioxidant and apoptosis gene expressions were detected to screen the target strain. Nuclear factor erythroid-derived 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein1 (Keap1) pathway, reactive oxygen species (ROS) production, mitochondrial membrane potential (Delta psi m), apoptosis, and necrosis were analyzed. Results revealed that heme oxygenase-1 (HO-1) gene expression had a positive correlation with H2O2 induction. Moreover, Bacillus amyloliquefaciens SC06 (SC06)-meditated IPEC-1 showed the best antioxidant capacity though modulating Nrf2 phosphorylation. Delta psi m was elevated, while ROS generation was reduced with SC06 pre-protection, resulting in decreased apoptosis and necrosis. Altogether, HO-1 expression could be regarded as an OS indicator. The regulation of Nrf2/Keap1 pathway and ROS production by SC06 are involved in alleviating OS of IPEC-1.