Background <p>The pathogenesis of bovine mastitis involves inflammation and cell death, with pyroptosis being a key factor in its development. Currently, antibiotics remain the primary therapeutic option for bovine mastitis, however, the increasing prevalence of antibiotic resistance constitutes a major public health threat. Selenium (Se) has been reported to alleviate inflammation primarily through its antioxidant properties, but the mechanism by which Se regulates pyroptosis in bovine mastitis remains unclear.</p> Methods and results <p>In this study, an in vitro mastitis model was established by infecting MAC-T cells with inactivated <i>Staphylococcus aureus</i> (<i>S. aureus</i>) (MOI = 10, 12&#xa0;h). The results revealed that the mitochondrial membrane potential of the MAC-T cells in the infection group decreased significantly. Moreover, the accumulation of Reactive oxygen species (ROS) was accompanied by the activation of NOD-like receptor family containing pyrin domain 3 (NLRP3), the expression of the pyroptosis-related genes gasdermin D amino terminal fragment (GSDMD-N), and cysteine ​​aspartate specific protease 1 (cleaved-caspase 1). Pretreatment with Se, the NLRP3 inhibitor MCC950 and the antioxidant N-acetylcysteine ​​(NAC) attenuated mitochondrial damage, ROS accumulation, and the inhibition of pyroptosis. An in vivo mastitis model was established in mice fed a high-selenium diet (containing 1.5&#xa0;mg/kg Se) and intramammarily injected with inactivated <i>S. aureus</i> (1 × 10⁸ CFU/mL). Histological analysis revealed intact alveolar structure and reduced inflammatory cell infiltration in mice fed the high-selenium diet. The expression of the inflammasome NLRP3 downregulated, and the expression of GSDMD-N, a direct executor of pyroptosis, upregulated.</p> Conclusion <p>Our in vitro and in vivo results confirmed that Se alleviate mitochondrial damage and pyroptosis by inhibiting the NLRP3 pathway, ultimately alleviating mastitis.</p> Graphical Abstract <p></p>