In vivo tests indicated that LCZ significantly reduced the injury and histological score of mice mammary tissues in E. coli-induced mastitis (p < 0.01) by significantly promoting the expression of the tight junction proteins claudin-3, occludin, and ZO-1 (p < 0.01), which ameliorated blood-milk barrier disruption, and decreasing the expression of the inflammatory cytokines (TNF-α, IL-1β, and IL-6) in mice mammary tissue (p < 0.01). This evidence concerns the gene CLDN3 and mastitis.