Purpose Estrogen plays a critical role in the invasiveness and metastasis of non-small cell lung cancer (NSCLC) through estrogen receptor beta (ER beta). However, the antimetastatic effect of the ER beta antagonist fulvestrant was still limited in NSCLC patients. Recently, Toll-like receptor 4 (TLR4) signaling was implicated in NSCLC metastasis. Our present study aimed to evaluate the synergistic antimetastatic effect of a combination of fulvestrant and the TLR4-specific inhibitor CLI-095 (TAK-242) on human NSCLC cells. Methods The expression levels of ER beta and TLR4 were detected by immunohistochemical (IHC) analysis of 180 primary NSCLC and 30 corresponding metastatic lymph node samples. The association between ER beta and TLR4 expression was analyzed. The aggressiveness of NSCLC cells treated with fulvestrant, CLI-095 or the drug combination and formation status of their invadopodia, invasion-associated structures, were investigated. The protein levels in NSCLC cells in different groups were determined by Western blot and immunofluorescence analyses. Results Here, a positive correlation between ER beta and TLR4 expression was observed in both primary NSCLC tissue (Spearman's Rho correlation coefficient = 0.411, p < 0.001) and metastatic lymph node tissue (Spearman's Rho correlation coefficient = 0.374, p = 0.009). The protein levels of ER beta in NSCLC cell lines were decreased by fulvestrant, and this suppressive effect was significantly enhanced when fulvestrant was combined with CLI-095 (p < 0.05). Both the migration and invasion of NSCLC cells were suppressed by fulvestrant or CLI-095 alone, and the combination of fulvestrant + CLI-095 showed the strongest inhibitory effect (p < 0.05). In addition, the results demonstrated that CLI-095 also helped fulvestrant restrict the formation and function of invadopodia in NSCLC cells (p < 0.05). Conclusions Collectively, our study results suggested that CLI-095 enhances the antimetastatic effect of fulvestrant on NSCLC and provided support for further investigation of the antitumor activity of combined therapy with antiestrogen and anti-TLR4 agents in the clinic.