The hormone prolactin (PRL) regulates differentiation and lactation in the bovine mammary epithelium. This tissue is especially prone to contracting mastitis, a disease characterized by an inflammatory response in the mammary gland. Staphylococcus aureus is the infectious agent primarily responsible for mastitis. In a previous study, we have shown that bovine PRL (bPRL) stimulates S. aureus internalization in bovine mammary epithelial cells (bMECs) by regulating several host innate immune elements, which are often modulated by nuclear factor kappa B (NF-κB). However, it is unknown whether the activation of the NF-κB transcription factor is regulated by bPRL during S. aureus internalization. The objective of this study was to determine the role of NF-κB in bPRL-stimulated bMECs during S. aureus internalization. Our results showed that bPRL (5 ng/ml) induced NF-κB activation in bMECs; however, it was inhibited by S. aureus in presence of the hormone. When we blocked NF-κB activation with acetylsalicylic acid, we detected an inhibition in S. aureus internalization (48%) in bPRL-stimulated bMECs. The infection-induced inhibition of NF-κB activation in the presence of bPRL correlates with the downregulation in bPRL-mediated tumor necrosis factor (TNF)-α (27%) and tracheal antimicrobial peptide (TAP, 70%) mRNA expression and nitric oxide (NO) production in bMECs. We also detected an inhibition in the expression of the bPRL target gene κ-casein (50%) under these conditions. Interestingly, these effects are not achieved through increased PRL receptor expression (PRLR), as it was inhibited (48%) compared to control cells. In conclusion, NF-κB activation in bMECs is inhibited by S. aureus in the presence of bPRL, suggesting a mechanism by which the host innate immune response may be compromised during subclinical mastitis.