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Cow mastitis, caused by Streptococcus infection of the mammary glands, is a common clinical disease that can lead to decreased milk quality and threaten animal welfare and performance. Esculetin (ESC) is a coumarin with anti-inflammatory and anti-asthmatic effects. However, whether ESC has therapeutic effects on mastitis remains unexplored. This study was conducted to investigate the protective effect of ESC against murine mastitis caused by Streptococcus isolated from bovine mammary glands and elucidate the underlying mechanisms. Streptococcus uberis was used to construct a mouse model of mastitis. The results showed that the mice exhibited edema and thickening of the acinar wall with inflammatory infiltration after S. uberis treatment. Intraperitoneal injection of ESC significantly reduced inflammatory cell infiltration, restored normal physiological function, and inhibited the production of the inflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analysis revealed that ESC reduced P38 phosphorylation, further inhibited the influence of mammary Streptococcus on cytoplasmic translocation of nuclear factor-κB (P65), and inhibited the transcriptional activation of P65, thus inhibiting the generation of inflammatory cells. Collectively, ESC may inhibit mitogen-activated protein kinase and nuclear factor-κB, thereby highlighting its potential for the treatment and prevention of mastitis. • Mastitis can lead to decreased milk quality, threatening animal welfare and performance, and affecting human health. • A mouse model of mastitis was constructed using Streptococcus mammary of bovine origin. • The natural herb esculetin protects against mastitis in mice caused by streptococcus isolated from bovine mammary glands. [ABSTRACT FROM AUTHOR]