Panax ginseng extract (PGe) has been shown to possess immunomodulatory effects in healthy dairy cows at drying off and to trigger an adequate immune response to protect from an experimental intramammary infection (IMI) with Staphylococcus aureus in a murine model. S. aureus is one of the major pathogens isolated from bovine IMI; being capable to invade and survive within mammary epithelial cells. However, the precise mechanism by which PGe interacts with bovine mammary epithelial cells (MAC-T) and bovine macrophages in the course of a S. aureus infection remains unclear. We evaluated the effect of PGe on MAC-T cytokine response and on the internalization of S. aureus into MAC-T. In addition, we evaluated the effect of PGe on the phagocytic activity of macrophages isolated from bovine mammary secretions. Results shown that MAC-T cells TLR4 and NF-κB mRNA expression was not affected by PGe at all evaluated times. IL-6 mRNA expression and protein level and IL-4 protein level were significantly induced in MAC-T treated with 3 mg/ml of PGe. PGe at 3 mg/ml reduced significantly the internalization of two S. aureus strains in MAC-T. In addition, PGe did not affect the percentage of phagocytosis and the NO and ROS production of macrophages co-cultured with two strains of S. aureus. These results, obtained in in vitro models together with those obtained in in vivo previous studies carried out in bovines and mice can contribute to improve the understanding of the effects of PGe following inoculation in bovine mammary glands.