Different inflammatory diseases in dairy cows are related to the impairment of neutrophils function, which is affected by blood oestrogen level. In this study, the molecular mechanism of 17β-oestradiol (E2) receptors mediated by the basal physiological concentration of E2 in the immune activity and energy status of circulating cow neutrophils was elucidated in vitro based on 9 blood samples collected from 3 adult Simmental cows at 2-weeks intervals. Western blotting and qRT‒PCR were used to investigate E2 nuclear receptors (ERα and ERβ) and G-protein coupled receptor (GPER) expression in resting and lipopolysaccharide (LPS)-activated neutrophils at the basal physiological level of E2 (20 pg/mL) in vitro and the effect on the inflammatory signaling pathway Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NFκB) and the AMP-activated protein kinase (AMPK), the regulators of energy metabolism. Results showed that E2 upregulated GPER protein expression, and decreased TLR4 mRNA and protein expression, myeloid differentiation Factor 88 (MyD88) mRNA expression and NFκB (p65) phosphorylation, but ERα and ERβ mRNA and protein expression were not changed. However, ERα mRNA and protein expression and GPER protein expression were decreased, and ERβ mRNA and protein expression was increased in activated neutrophils. TLR4 mRNA and protein expression, MyD88 mRNA expression and NFκB (p65) phosphorylation were upregulated, while AMPK phosphorylation was inhibited in activated neutrophils, with an opposite effect on oestrogen-treated neutrophils. In conclusion, basal physiological E2 concentration can mediate ERα, ERβ and GPER to slow down the activation of the TLR4/NFκB signaling pathway in LPS-activated neutrophils and to promote cell energy metabolism in cows.