Tumor cell expression of Toll-like receptors (TLRs) can promote inflammation and cell survival in the tumor microenvironment. Toll-like receptor 4 (TLR4) signaling in tumor cells can mediate tumor cell immune escape and tumor progression, and it is regarded as one of the mechanisms for chronic inflammation in tumorigenesis and progression. The expression of TLR4 in human breast cancer cell line MDA-MB-231 and its biological function in the development and progression of breast cancer have not been investigated. We sought to characterize the expression of TLR1-TLR10 in the established human breast cancer cell line MDA-MB-231, and to investigate the biological roles of TLR4 in breast cancer cells growth, survival, and its potential as a target for breast cancer therapy. TLRs mRNA and protein expressions were detected in human breast cancer cell line MDA-MB-231 by RT-PCR, real-time PCR and flow cytometry (FCM). RNA interference was used to knockdown the expression of TLR4 in MDA-MB-231. MDA-MB-231 transfected with the vector pGenesil-1 and the vector containing a scrambled siRNA were as controls. Recombinant plasmids named TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA specific to TLR4 were transfected into human breast cancer cell line MDA-MB-231 with Lipfectamine 2000 reagent. TLR4 mRNA and protein expressions were investigated by RT-PCR, real-time PCR, FCM and immunofluorescence after silence. MTT analysis was performed to detect cell proliferation and FCM was used to detect the secretion of inflammatory cytokines in supernatant of transfected cells. The human breast cancer cell line MDA-MB-231 was found to express TLR1-TLR10 at both the mRNA and protein levels. TLR4 was found to be the highest expressed TLR in MDA-MB-231. TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA were found to significantly inhibit TLR4 expression in MDA-MB-231 at both mRNA and protein levels as compared to vector control(vector transfected cells). TLR4AsiRNA mediated the strongest effect. Knockdown of TLR4 gene in MDA-MB-231 resulted in a dramatic reduction of breast cancer cell viability. The cytokines which were secreted by the TLR4 silenced cells, such as IL-6 and IL-8, also decreased significantly as compared with vector control. No significant difference was observed in siRNA control (Recombinant plasmid named ScrambledsiRNA transfected cells) compared to vector control. These studies identified the expression levels of multiple TLRs in human breast cancer cell line MDA-MB-231 and demonstrated that knockdown of TLR4 could actively inhibit proliferation and survival of breast cancer cells. Taken together, our results suggest RNAi-directed targeting of TLR4 may be a beneficial strategy for breast cancer therapy.