Mastitis is a frequent disease of dairy cows and results in significant economic losses for dairy producers. In this study, Establishment of inflammation was performed by using different concentrations of lipopolysaccharide (LPS). After that, mammary alveolar cell-T (MAC-T) were randomly cultured in the standard medium (CK), standard medium with 100 μg/mL LPS (LPS₁₀₀), standard medium with 10 mmol/L rapamycin (RAP₁₀), and standard medium with 100 μg/mL LPS＋10 mmol/L RAP (LPS₁₀₀＋RAP₁₀) for 24 h. Cells and culture supernatant were collected at the end of the treatment. The results showed that incubation with LPS for 24 h significantly increased the concentrations of interleukin-8 (IL-8), interleukin-1β (IL-1β) and tumor necrosis factor- α (TNF- α) in MAC-T cells (P＜0.05) without affecting the cell viability and apoptosis. Rapamycin addition individually had no significant effects on baseline concentrations of IL-1β and TNF-α (P＞0.05), but abolished the increased production of inflammatory cytokines stimulated by LPS (P＜0.05). Culturing with LPS increased the phosphorylation and translocation of p65 protein in nuclear factor-κB (NF-κB) signaling pathway, while this elevation was disappeared with the rapamycin supplementation (P＜0.05). Phosphorylation levels of c-Jun N-terminal kinase (JNK) and p38 proteins in mitogen-activated protein kinase (MAPK) signaling pathway were lower in LPS₁₀₀＋RAP₁₀ group than that in LPS₁₀₀ group (P＜0.05). These results indicate that rapamycin alleviates LPS-induces inflammatory response in MAC-T cells through the NF-κB/MAPK pathway, which gives a reference for the therapeutic potential of rapamycin in mastitis.