Toll-like receptor 4 (TLR4)-mediated signaling has been implicated in tumor cell invasion, survival and metastasis in several types of cancers. However, the expression of TLR4 in patients with non-small cell lung cancer (NSCLC) and its biological function in the development and progression of NSCLC have not been elucidated to date. Here, we sought to characterize the expression of TLR4 in patients with NSCLC and to investigate the biological roles of TLR4 in lung metastasis, cell invasion and survival. In this study, we found that TLR4 expression was elevated in most patients with NSCLC, and its expression levels correlated with key pathological characteristics, including tumor differentiation, stage and metastasis. Our data also showed that downregulation of TLR4 expression using an RNA silencing approach in A549 tumor cells significantly suppressed cell proliferation, cell migration and cell invasion, and induced tumor apoptosis in vitro, and suppressed tumor growth in vivo. In addition, we also found that downregulation of TLR4 expression significantly decreased cell TNF-α and IL-6 levels. Furthermore, we found that knockdown of TLR4 was able to significantly suppress constitutive phosphorylation of Akt and PI3K, which may contribute to the inhibition of tumor growth. These data suggest that TLR4 plays an important role in tumorigenic properties of human NSCLC, and that RNA interference-directed targeting of TLR4 could be used as a potential anticancer therapeutic target in NSCLC.