The objectives of this study were to investigate the effect of Escherichia coli lipopolysaccharide (LPS) on the function of bovine neutrophils (PMNL) collected from mid lactation cows and determine the differential effects of LPS on gene expression of PMNL purified from early and mid lactation cows. The PMNL from mid lactation cows (187 ± 13 d postpartum) were incubated with 0, 1, 25, and 50 μg/mL of LPS for 120. min, and the generation of reactive oxygen species (ROS), PMNL extracellular traps (NET), chemotaxis, and killing of Staphylococcus aureus were determined. Incubation of PMNL with 25 μg/mL of LPS increased intracellular ROS by 79% in mitogen-stimulated PMNL. Addition of 50 μg/mL of LPS enhanced intracellular ROS by nonstimulated and stimulated PMNL by 184 and 154%, respectively. Nonstimulated PMNL incubated with 25 and 50 μg/mL of LPS had a 105% increase in NET. Addition of LPS had no effect on subsequent PMNL chemotaxis or killing of Staph. aureus. To examine the effect of LPS on the expression of genes involved in PMNL function and cytokine production, mRNA was purified from PMNL isolated from mid lactation (146 ± 2 postpartum; n = 10) and early lactation cows (7 d postpartum; n = 10), after a 120-min incubation with 0 or 50 μg/mL of LPS. Amounts of interleukin-8 (IL-8), tumor necrosis factor (TNF), bactericidal/permeability-increasing protein (BPI), myeloperoxidase (MPO), superoxide dismutase 2 (SOD2), NADPH oxidase 4 (NOX4), Cytochrome b-245, α polypeptide (CYBA), histone H2A/1 (H2A/1), and histone H2B-like (H2B) mRNA were determined relative to that of β-actin by real-time quantitative PCR. Regardless of stage of lactation, PMNL incubated with 50 μg/mL of LPS had 537 and 45% higher mRNA contents of IL-8 and SOD2 compared with 0 μg/mL LPS, respectively. In addition, LPS augmented the expression of TNF, BPI, and CYBA (2,908, 59, and 158% compared with controls, respectively) only in PMNL from mid lactation cows. Addition of LPS did not affect mRNA levels of MPO, NOX4, H2A/1, or H2B. Independent of LPS treatment, PMNL from mid lactation cows had 99% higher mRNA contents of IL-8 compared with PMNL from early lactation cows. The PMNL from early lactation cows had a 634% increase in MPO mRNA expression relative to that from mid lactation cows. These results support that LPS directly stimulates PMNL to produce ROS and express NET. In addition, LPS enhances the generation of ROS by PMNL in response to other stimuli and increases the expression of genes encoding inflammatory mediators and enzymes involved in the production of ROS. Finally, reduced PMNL gene expression of IL-8 (regardless of LPS activation), TNF, CYBA, and BPI (upon stimulation with LPS) in early lactation may elucidate several mechanisms by which PMNL may become immune-incompetent during this period.