High incidence of traditional and emerging Fusarium mycotoxins in cereal grains and silages can be a potential threat to feed safety and ruminants. Inadequate biodegradation of Fusarium mycotoxins by rumen microflora following ingestion of mycotoxin-contaminated feeds can lead to their circulatory transport to target tissues such as mammary gland. The bovine udder plays a pivotal role in maintaining milk yield and composition, thus, human health. However, toxic effects of Fusarium mycotoxins on bovine mammary gland are rarely studied. In this study, the bovine mammary epithelial cell line was used as an in-vitro model of bovine mammary epithelium to investigate effects of deoxynivalenol (DON), enniatin B (ENB) and beauvericin (BEA) on bovine mammary gland homeostasis. Results indicated that exposure to DON, ENB and BEA for 48 h significantly decreased cell viability in a concentration-dependent manner (P< 0.001). Exposure to DON at 0.39 μmol/L and BEA at 2.5 μmol/L for 48 h also decreased paracellular flux of FITC-40 kDa dextran (P< 0.05), whereas none of the mycotoxins affected transepithelial electrical resistance after 48 h exposure. The qPCR was performed for assessment of expression of gene coding tight junction (TJ) proteins, toll-like receptor 4 (TLR4) and cytokines after 4, 24 and 48 h of exposure. DON, ENB and BEA significantly upregulated the TJ protein zonula occludens-1, whereas markedly downregulated claudin 3 (P< 0.05). Exposure to DON at 1.35 μmol/L for 4 h significantly increased expression of occludin (P< 0.01). DON, ENB and BEA significant downregulated TLR4 (P< 0.05). In contrast, ENB markedly increased expression of cytokines interleukin-6 (IL-6) (P< 0.001), tumor necrosis factor a (TNF-a) (P< 0.05) and transforming growth factor-β(TGF-β) (P< 0.01). BEA significantly upregulated IL-6 (P< 0.001) and TGF-β (P= 0.01), but downregulated TNF-α (P< 0.001). These results suggest that DON, ENB and BEA can disrupt mammary gland homeostasis by inducing cell death as well as altering its paracellular permeability and expression of genes involved in innate immune function.