Abstract The role of regulatory T cells (T regs) is well documented in immune homeostasis and protection against autoimmune disease. Forkhead box protein 3 (FOXP3) has been shown to be essential for the development and function of T reg. Due to the lack of tools for FOXP3 detection in certain species, understanding the role of T reg in a variety of ruminant diseases has been hampered. In this study, we developed monoclonal antibodies (mAbs) against bovine FOXP3 using recombinant bovine FOXP3 lacking the forkhead domain as an immunogen. The specificity of the mAbs was confirmed by immunoblot and mass spectrometry. Expression of FOXP3 was induced in bovine PBMCs after 6 d of exposure to staphylococcal enterotoxin type C1 (SEC1) in vitro. Similar to findings in mice and humans, expression of FOXP3 was restricted to CD4 + CD25 + T cells. Transcriptional analysis of bovine TCR variable regions of the β chain (boVβ) showed that transcription of boVβ sequences reactive with SEC1 increased for 6 d, and then boVβ sequences non-reactive with SEC1 rapidly increased in the cultures. This indicates that induction of FOXP3 + CD4 + CD25 + T regs by SEC1 is not Vβ restricted. The FOXP3 mAbs developed in this study will be useful in the further investigation of the role of T reg in staphylococcal pathogenesis in bovine mastitis and other ruminant diseases.