Staphylococcus aureus, a major pathogen for the mammary gland of dairy ruminants, elicits the recruitment of neutrophils into milk during mastitis, but the mechanisms are incompletely understood. We investigated the response of the bovine mammary gland to muramyl dipeptide (MDP), an elementary constituent of the bacterial peptidoglycan, alone or in combination with lipoteichoic acid (LTA), another staphylococcal microbial-associated molecular pattern (MAMP). MDP induced a prompt and marked influx of neutrophils in milk, and its combination with LTA elicited a more intense and prolonged influx than the responses to either stimulus alone. The concentrations of several chemoattractants for neutrophils (CXCL1, CXCL2, CXCL3, CXCL8, and C5a) increased in milk after challenge, and the highest increases followed challenge with the combination of MDP and LTA. MDP and LTA were also synergistic in inducing in vitro chemokine production by bovine mammary epithelial cells (bMEpC). Nucleotide-binding oligomerization domain 2 (NOD2), a major sensor of MDP, was expressed (mRNA) in bovine mammary tissue and by bMEpC in culture. The production of interleukin-8 (IL-8) following the stimulation of bMEpC by LTA and MDP was dependent on the activation of NF-κB. LTA-induced IL-8 production did not depend on platelet-activating factor receptor (PAFR), as the PAFR antagonist WEB2086 was without effect. In contrast, bMEpC and mammary tissue are known to express Toll-like receptor 2 (TLR2) and to respond to TLR2 agonists. Although the levels of expression of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β were increased by LTA and MDP at the mRNA level, no protein could be detected in the bMEpC culture supernatant. The level of induction of IL-6 was low at both the mRNA and protein levels. These results indicate that MDP and LTA exert synergistic effects to induce neutrophilic inflammation in the mammary gland. These results also show that bMEpC could contribute to the inflammatory response by recognizing LTA and MDP and secreting chemokines but not proinflammatory cytokines. Overall, this study indicates that the TLR2 and NOD2 pathways could cooperate to trigger an innate immune response to S. aureus mastitis.