Oxidative stress causes damage to macromolecules, including proteins, DNA, and lipid, and has been recognized as a crucial driver of the onset and progression of several intestinal disorders. Pterostilbene, one of the natural antioxidants, has attracted considerable attention owing to its multiple biological activities. In the present study, we established an oxidative stress model in broiler chickens via injection with diquat to investigate whether pterostilbene could attenuate diquat-induced intestinal damage and reveal the underlying mechanisms. We found that diquat-induced decreases in the activities of superoxide dismutase and glutathione peroxidase and the level of reduced glutathione and the increase in hydrogen peroxide content in plasma and jejunum were significantly alleviated by pterostilbene ( P < 0.05 ). Pterostilbene supplementation also decreased intestinal permeability and jejunal apoptosis rate, improved jejunal villus height and the ratio of villus height to crypt depth, and promoted the transcription and translation of jejunal tight junction proteins occludin and zona occludens 1 in diquat-challenged broilers ( P < 0.05 ). Furthermore, pterostilbene reversed diquat-induced mitochondrial injury in the jejunum, as indicated by the decreased reactive oxygen species level and elevated activities of superoxide dismutase 2 and mitochondrial respiratory complexes ( P < 0.05 ). Importantly, administering pterostilbene maintained iron homeostasis, inhibited lipid peroxidation, and regulated the expression of the markers of ferroptosis in the jejunum of diquat-exposed broilers ( P < 0.05 ). The nuclear factor erythroid 2-related factor 2 signaling pathway in the jejunum of diquat-exposed broilers was also activated by pterostilbene ( P < 0.05 ). In conclusion, our study provides evidence that pterostilbene alleviates diquat-induced intestinal mucosa injury and barrier dysfunction by strengthening antioxidant capacity and regulating ferroptosis of broiler chickens.