Background and Purpose: Escherichia coli is a common mastitis-causing pathogens and is destructive to the blood-milk barrier. Oral administration of Lactobacillus casei Zhang (LCZ) could alleviate mice mastitis. However, its prophylactic effect and mechanism through intramammary injection on E. coli-induced mastitis is unclear now. Here, we investigate this using E. coli-induced mastitis model. Experimental Approach: Prophylactic effects and mechanism of intramammary injection of LCZ on blood-milk barrier and inflammation were studied in both bovine mammary epithelial cells (BMECs) and pregnant mice. Key Results: In vitro tests revealed that LCZ significantly inhibited the adhesion of E. coli to monolayer cells, reduce the damage of cell desmosomes, up-regulated the expression of tight junction proteins (claudin-1, claudin-4, occludin, and ZO-1), and down-regulated the expression of inflammatory cytokines (TNF-α, IL-1β, and IL-6) thereby enhancing the trans-epithelial electric resistance of monolayer BMECs and effectively protecting cells from damage caused by E. coli. In vivo experiments suggested that LCZ significantly promoted the expression of tight junction proteins (claudin-3, occludin, and ZO-1) but significantly inhibited the expression of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in mouse mammary tissue, thereby decreased disruption in mammary tissues, infiltration of inflammatory cells in E. coli-induced mastitis. Conclusions and Implications: In our study, LCZ ameliorates blood-milk barrier disruption and suppresses the inflammatory response during E. coli-induced mastitis, indicating LCZ may serve as a effective prophylactic agent to preserve the blood-milk barrier function during mastitis.