Bovine mastitis is defined as the inflammation of mammary gland and is the most multiple diseases in dairy cattle. There is still no effective treatment now. Leonurine, extracted from Leonurus cardiaca, has been proved to have anti-inflammatory effect. In the present study, we utilized a mouse mastitis model to study the effect of leonurine on LPS-induced mastitis. Leonurine was administered three times during the 24 h after inducing infection in the mammary gland. The results showed that leonurine significantly alleviated LPS-induced histopathological changes, downregulated the levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), upregulated the level of anti-inflammatory cytokine interleukin-10 (IL-10), and inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Further study revealed that leonurine inhibited the expression of Toll-like receptor 4 (TLR4) and the activation of nuclear factor-kappaB (NF-κB) and the phosphorylation of p38, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK). Therefore, the results demonstrated that leonurine could downregulate the expression of TNF-α, IL-6, iNOS, and COX-2 and upregulate the expression of IL-10 mainly by inhibiting the expression of TLR4 and the activation of NF-κB and the phosphorylation of p38, ERK, and JNK. Leonurine may be a potential agent for mastitis therapy.