Lactic acid bacteria (LAB) play a key role in promoting health and preventing diseases because of their beneficial effects, such as antimicrobial activities, modulating immune responses, maintaining the gut epithelial barrier and antioxidant capacity. However, the mechanisms with which LAB relieve oxidative stress and intestinal injury induced by diquat in vivo are poorly understood. In the present study, Pediococcus pentosaceus ZJUAF-4 (LAB, ZJUAF-4), a selected probiotics strain with strong antioxidant capacities, was appointed to evaluate the efficiency against oxidative stress in diquat-induced intestinal injury of mice. Alanine transaminase (ALT) and aspartate aminotransferase (AST) were analyzed to estimate the liver injury. The intestinal permeability was evaluated by 4 kDa fluorescein isothiocyanate (FITC)-dextran (FD4), D-lactate (DLA), and diamine oxidase (DAO) levels. Jejunum reactive oxygen species (ROS) production was examined by dihydroethidium (DHE) staining. Western blotting was used to detect the expression of nuclear factor (erythroid-derived-2)-like 2 (Nrf2) and its downstream genes in jejunum. The gut microbiota was analyzed by high-throughput sequencing method based on the 16S rRNA genes. The results showed that ZJUAF-4 pretreatment was found to protect the intestinal barrier function and maintain intestinal redox homeostasis under diquat stimulation. Moreover, oral administration of ZJUAF-4 increased the expression of Nrf2 and its downstream genes. High-throughput sequencing analysis indicated that ZJUAF-4 contributed to restoring the gut microbiota influenced by diquat. Our results suggested that ZJUAF-4 protected the intestinal barrier from oxidative stress-induced damage by modulating the Nrf2 pathway and gut microbiota, indicating that ZJUAF-4 may have potential applications in preventing and treating oxidative stress-related intestinal diseases.