Recently, we observed that the application of Escherichia coli lipopolysaccharide (LPS) into the mammary gland induce severe clinical symptoms (pyrexia, increased cardiac and respiratory frequencies), an intense stress, and systemic immune response (increased plasma cortisol and haptoglobin concentrations). However, no obvious suppression of luteal size, blood flow, and progesterone secretion in lactating dairy cows were detectable. To investigate possible effects of intramammary LPS challenge on gene expression in luteal tissue, biopsies of the corpus luteum (CL) were analyzed. On d 9 of the estrous cycle (d 1 = ovulation), 8 lactating dairy cows received once 200 µg LPS (dissolved in 10 mL NaCL) and 6 cows received once 10 mL saline (control) into one quarter of the mammary gland. Luteal tissue was collected for biopsy 24 h before and 6 h after treatment, using an ultrasound-guided semi-automatic high-speed biopsy needle. RT-qPCR was applied to assess the mRNA expression of toll-like receptors (TLR2, TLR-4), tumor necrosis factor α (TNFα), steroidogenic factors (STAR, 3βHSD), and factors related to apoptosis (CASP3) and prostaglandin synthesis (PGES, PGFS, FP). Data were normally distributed (Shapiro-Wilk-test) and Student's t-test was used for dependent comparisons of repeated measures. LPS challenge increased (P ≤ 0.05) mRNA abundance of TNFα, TLR2 and CASP3, whereas TLR4, STAR, 3βHSD, and prostaglandin-related factors did not change significantly. In control cows, only mRNA expression of CASP3 was increased (P ≤ 0.05) at 6 h after compared with 24 h before treatment. Our results indicate that LPS-induced mammary immune response increases luteal TNFα and TLR2 synthesis but do not induce luteolysis in the bovine CL.