The endometrium is easily contaminated with bacteria and the endometrial epithelial cells (EECs) play an important role in defence against invading pathogens which recognized pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs).Toll-like receptor 4 (TLR4) can recognize lipopolysaccharide (LPS) from Gram-negative bacteria and initiates innate immune responses.In this study, we stimulated bovine EECs with LPS from Escherichia colt (E.colt).The expression of TLR4 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot.The expression of downstream TLR4 signaling molecules was detected by qRT-PCR.The results showed that TLR4 and downstream adaptor molecules, transcription factors and cytokines were up-regulated when bovine EECs were stimulated with LPS.Furthermore, the expression of TOLLIP and β-defensin 5 were up-regulated when cells were stimulated with LPS.The results demonstrated that both MyD88 dependent and independent pathways in TLR4 were activated by LPS in bovine EECs.Bovine EECs have the immune repertoires required in defending against E.colt and play an important role in innate immune defence of the bovine endometrium.