Estrogen promotes non-small cell lung cancer (NSCLC) metastasis via estrogen receptor beta (ER beta)-mediated invasiveness-associated matrix metalloprotease 2 (MMP2) upregulation. However, how ER beta increases the aggressiveness of NSCLC cells remains unclear. Recently, MMP2 was found to be upregulated by Toll-like receptor 4 (TLR4) signaling activation and to promote NSCLC metastasis. Our present study aimed to examine the role of ER beta in the activation of TLR4 signaling and in tumor progression and metastasis, and to explore the synergistic metastatic effect of a combination of ER beta and TLR4 activation on human NSCLC cells in vitro and in vivo. Here, we found that ER beta is associated with TLR4 in metastatic lymph nodes. Western blot analysis and immunofluorescence revealed that ER beta overexpression upregulated TLR4 protein expression and activated downstream targets, myeloid differentiation primary response 88 (myd88)/nuclear factor (NF)-kappa B/MMP2, enhancing NSCLC cell migration and invasion in vitro. A novel ER beta-TLR4 interaction in cell plasma was identified by co-immunoprecipitation and confocal immunofluorescence. The combination of estradiol and specific TLR4 agonist lipopolysaccharide (LPS) synergistically promoted metastatic behaviors in NSCLC cells. In cell culture and murine lung metastasis models, exposure to estradiol and LPS induced increased matrix degradation and accelerated invadopodia and metastasis formation in NSCLC cells compared with that in cells treated with estradiol or LPS alone. Together, we showed that estrogen promoted NSCLC metastasis via ER beta by upregulating TLR4 and activating its downstream signaling axis myd88/NF-kappa B/MMP2. The combined targeting of ER beta and TLR4 may be a novel therapeutic strategy against advanced metastatic lung cancer.