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We proposed in this study that during intramammary infection, biofilm formation may facilitate adherence and colonization of Enterococcus faecalis to mammary gland epithelium. This was established by comparing six different Ent. faecalis isolates with different biofilm-forming profiles for their adhesive, invasive and survival capabilities to bovine mammary epithelial cell line (MAC-T). Our results showed increased ability of the biofilm-producer Ent. faecalis strains to adhere, invade and survive inside MAC-T cells rather than nonbiofilm-producer strains. We showed that growth of bacteria in bovine milk significantly augmented the adherence and invasion of all tested strains, and this feature was abolished again when strains were subcultured in brain heart infusion broth. Moreover, growth in bovine milk significantly increased biofilm formation by all tested strains. These results indicated that biofilm formation by Ent. faecalis, especially after expressing milk-dependent induction, may have special relevance in the pathogenesis of Ent. faecalis mastitis during intramammary infection by enhancing bovine mammary epithelial adhesion and colonization.Results obtained from current work highlighted the role of biofilm in the pathogenesis of Enterococcus faecalis mastitis. Those biofilm-forming strains might be substantial as useful antigens in diagnostic assays and as future vaccine candidates to control Ent. faecalis mastitis.