Mastitis is an inflammation of the mammary gland commonly caused by bacterial infection. Mammary epithelial cells mount defense against invading pathogens by detecting their respective danger signals or ligands and initiating appropriate immune responses. Caveolaes are a subset of lipid rafts that are rich in glyco-sphingolipids and cholesterol, mediate nonclathrin-dependent endocytosis, and regulate the internalization of particles such as bacteria. Caveolin-1, component of caveolae membranes, has been implicated as a modulator of innate immunity and inflammation. TLRs, Toll-like receptors, play central roles in the regulation of the host immune system and each TLR recognizes specific pathogen-associated molecular patterns. TLR4 is one of the well characterized pathogen recognition receptors that recognizes the LPS of Gram-negative bacteria. To explore the role of caveolin-1 gene silencing on mitogen-activated protein kinase (MAPK) activation in lipopolysaccharide (LPS)-challenged human mammary epithelial cells, we established MCF-10ACE of caveolin-1 gene silencing from human mammary epithelial cell line MCF-10A by iRNA technology. DNA micro-assay was used to detect the expression file of inflammation-associated genes in MCF10ACE. Western blot analysis was used to examine the activation of mitogen-activated protein kinase (MAPK) in lipopolysaccharide (LPS)-challenged MCF-10A and MCF-10ACE. Moreover, immunofluorescence and Western blot analysis were performed to detect the co-localization of caveolin-1 and toll-like receptor 4 (TLR4) in human mammary epithelial cells. We found that MCF-10ACE exhibited significant increases in inflammation-associated genes expression, such as Prostaglandin-endoperoxide synthase 2 (PTGS2), B-cell lymphoma 2 (BCL2), Fas ligand (FAS) and Interleukin 2 receptor alpha (IL2Rα) were increased by >1.5-fold, even Interleukin 6 (IL-6) was increased by 7-fold and Interleukin 6 receptor (IL-6R) was increased by 17-fold.. In addition, LPS-induced p38 MAPK and JNK MAPK activation were significantly increased in MCF-10ACE (p<0.01). Furthermore, caveolin-1 colocalized with TLR4 and appeared a negative correlation trend. In conclusion, caveolin-1 gene silencing promotes MAPK activation via TLR4 signaling in human mammary epithelial cells response to LPS.