Mammary gland (MG) infections (mastitis) are frequent diseases of dairy cows that affect milk quality, animal welfare and farming profitability. These infections are commonly associated with the bacteria Escherichia coli and Staphylococcus aureus. Mastitis-causing pathogens invade the MG via the teat canal, then spread through the teat sinus, the gland cistern and the large ducts to disseminate in the MG. Different in vitro models have been used to investigate the early response of the MG to bacteria, but the role of the teat in mastitis pathogenesis has received less attention. This study is aimed at using punchexcised teat tissue as an ex vivo model to explore the immune mechanisms that arise early during MG infections. Teats were collected from MGs with no signs of disease from 28 slaughtered dairy cows in a French commercial abattoir. Teats were dissected under sterile conditions and explants (about 50 mg) were collected from the mucosal surface of teat sinus using sterile 6-mm-diameter biopsy punches. Explants were exposed to E. coli ultra-pure lipopolysaccharide (LPS), S. aureus lipoteichoic acid (LTA), as well as to heat-killed E. coli (strain P4) and S. aureus (D4 169-39 strain). Tissue viability and cellularity were evaluated by lactate dehydrogenase (LDH) release, microscopic examination and flow cytometry. Expression of proinflammatory mediators was evaluated at protein and mRNA levels by ELISA and qPCR, respectively. Cytotoxicity and microscopic analyses showed that bovine teat sinus explants have their morphology and viability preserved after ex vivo stimulation with TLR-agonists or bacteria for 24 h. Analysis of cell populations by flow cytometry revealed that the proportion of leukocytes, among them granulocytes and macrophages, is preserved after treatments. Regarding tissue immune response to stimulations, it could be observed that LPS and E. coli trigger stronger inflammation in teat when compared to LTA and S. aureus, leading to a higher production of IL-6 and IL-8, as well as to an up-regulation of proinflammatory genes such as IL1α , IL1β and TNFα. Ex vivo stimulation of punch-excised explants reproduced previous in vivo observations indicating that E. coli and its LPS cause stronger inflammatory response in the bovine MG when compared to S. aureus and LTA. Therefore, in compliance with the 3Rs principle (replacement, reduction, and refinement) in animal experimentation, ex vivo explant analyses proved to be a simple and affordable approach to study MG immune response to infection. The proposed model could be successfully based on slaughter industry waste and, contrary to sliced or chopped explants, showed as advantage keeping tissue architecture, with normal spatial arrangement of cells and interactions with extracellular matrix. This advantage might enable punch-excised explants to yield more biologically relevant responses to stimuli and better reproduce organ complexity than epithelial cell cultures or tissue slices. In summary, our model lends itself particularly well to studying the early phases of the MG immune response to infection might be of great use in future research on mastitis.