Mastitis often occurs during women’s lactation period, causing great psychological and physical pain to women. Fucoidan, a sulfated polysaccharide obtained from brown algae, has much broader biological properties. However, the roles of fucoidan in lipopolysaccharides (LPS)-induced mastitis are still undiscovered. The present study was aimed to evaluate the influences of fucoidan on LPS-induced mouse mastitis and investigate its possible mechanisms. The expression profiles of fucoidan acting on mastitis were analyzed by network pharmacology. Additionally, mechanism experiments verified the mechanism of fucoidan on mastitis. The results of in vivo study displayed that the treatment of fucoidan to LPS-stimulated mouse mastitis decreased the inflammatory damage, proinflammatory cytokines level and repaired the completeness of blood-milk barrier. In the study of mouse mammary epithelial cells, fucoidan suppressed the secretion of reactive oxygen species (ROS) and enhanced the activity of antioxidant enzymes. Molecular experiments suggested that fucoidan promoted nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear import and autophagy via activating the adenosine 5’-monophosphate-activated protein kinase (AMPK) signaling pathway. The above results indicated that in mastitis, fucoidan promoted Nrf2 nuclear import and autophagy via activating the AMPK signaling pathway, thereby suppressing oxidative stress and inflammatory injury to the mammary glands and protecting the blood-milk barrier.