Bovine mammary epithelial cells (BMECs) were successfully cultivated using tissue mass inoculation method in this study,and fibroblast cells was removed by adding nutrition factors and controlling the time of digestion.The isolated BMECs were thoroughly morphological and structural characteristic via immunocytochemistry, morphology, scanning electron microscopy (SEM) and transmission electron microscope (TEM).Our results showed that the obtained BMECs morphology together with the positive staining for Cytokeratin 18 was a powerful result to prove the specific epithelium characters of BMECs.The obtained BMECs were present as acobblestone form and proliferating monolayer.The cells had normal morphology, growth characteristics and mitochondrial and endoplasmic reticulum in cell cytoplasms, BMECs could be passaged 15 generations with a good behavior of proliferation, thus, it might represent an useful tool for studying the function of bovine mammary gland.Escherichia coli (E.coli) is the major pathogens causing mammary infection of dairy ruminants.Probiotics are extensively used to promote gastrointestinal health, and emerging evidence suggests that their beneficial properties can extend beyond the local environment of the gut.Here, we determined whether pre-incubated with probiotics can protect against E.coli-induced mastitis.In medium alone, the BMECs were plump and with plenty of microvilli, whereas E.coli infection caused microvilli missing, cellular atrophy and different degrees of cell collapse.In cells infected with E.coli alone, E.coli adhered on the surface of BMECs and the E.coli internalization in nucleus was observed.The BMECs incubated with only L.rhamnosus GR-1 maintained the intact ultrastructure and were with microvilli and plump surface.Moreover, pretreatment with L.rhamnosus GR-1 prevented disruption of the ultrastructure of BMECs for up to 6 h following E.coli challenge.In medium alone, the BMECs exhibited normal appearance (the cells with intact cytoplasm and organelles),including intact cytoplasm, plenty of microvilli and tight junctions (between two cells was often seen), the homogeneous electron density, integrated mitochondrial structures and endoplasmic reticulum structures in cell cytoplasm.However, E.coli infection caused microvilli rupture, loose cytoplasmic matrix structure and nuclear cavitation, hazy mitochondrial structures and degenerated into myeloid body.The BMECs incubated with only L.rhamnosus GR-1 maintained the normal ultrastructure compared with untreated BMEC control cells.Pretreatment with L.rhamnosus GR-1 prevented disruption of subcellular structure of BMECs for up to 6 h following E.coli challenge.Our results showed that pre-incubation with L.rhamnosus GR-1 ameliorated E.coli-induced disruption of BMEC morphology and ultrastructure at 6 h after challenge.In conclusion, our data indicate that a select L.rhamnosus strain interacts with BMECs to maintain the normal morphological structure during E.coli infection.These data could provide an important contribution to the development of an optimal regimen for preventing acute infection and understand the mechanism underlying probiotic maintaining the host healthy.