Infection of mammary glands by pathogenic bacteria causes inflammation and elicits structural and functional regression of mammary alveoli. The aim of the study was to evaluate quantitative changes in mammary alveolar structure, leukocyte infiltration into alveoli, and mammary apoptosis in response to infusion of endotoxin (lipopolysaccharide; LPS) in lactating cows. Ten multiparous cows, blocked by days in milk, parity and milk yield, were divided into treatment and control groups. In treatment (T) cows, both the front and rear quarters on the right or left side of the udder were assigned randomly to receive either LPS (50 µg in 10 mL, TL) or saline (10 mL, TS); the contralateral quarters received the other treatment. Udder-halves of control (C) cows were similarly assigned to receive either saline (10 mL, CS) or no infusion (untreated; CU). Mammary biopsies were obtained at 0 h (0600), 3 h (0900) and 12 h (1800) from each hind quarter of treatment and control cows. Endotoxin treatment induced 5.2- and 7.2- fold increases in number of neutrophils in alveolar lumina at 3 h and 12 h, respectively (P < 0.01), whereas neutrophils were rarely observed in the saline-infused control glands (TS and CS). Although the alveolar size was increased at 3 h and 12 h in comparison to 0 h (P < 0.05), it was not affected by LPS challenge. At 12 h, there was a decrease in the mean area stained positively for E-cadherin, an epithelial cell marker, in mammary glands treated with endotoxin (6.89 + 0.28% vs. 8.75 + 0.30%, or 8.1 + 0.29%, P < 0.01) for TL vs TS or CS, respectively. In addition, the percentage of cells stained with cleaved caspase-3, a marker of apoptosis, was increased by 3 h post-LPS infusion and showed significant time by treatment effects (15.9 + 0.92% vs. 8.1 + 0.92%; P < 0.01) for TL vs. TS. The majority of cleaved caspase-3-positive cells were stromal cells. These results demonstrate that LPS induces neutrophil infiltration into the alveolar lumen within 3 h, decreases E-cadherin expression, and promotes apoptosis in the mammary gland of lactating cows.