In non-small cell lung cancer (NSCLC), estrogen significantly promotes NSCLC cell growth via estrogen receptor beta (ER beta). However, the effects by which ER beta contributes to metastasis in NSCLC have not been previously reported. This study aims at defining whether the stimulation of ER beta promotes NSCLC metastasis in vitro and in vivo. Here, Our results showed that estrogen and ER beta agonist enhanced aggressiveness of two lung cancer cell lines (A549 and H1793) and promoted murine lung metastasis formation. ER-inhibitor Fulvestrant treatment or ER beta-knockdown significantly suppressed the migration, invasion and nodule formation of NSCLC cells. The expression level of ER beta protein was analyzed in matched samples of metastatic lymph node and primary tumor tissues from the same individuals, and we found significantly higher levels of ER beta were expressed in lymph node compared to primary tumor tissues. Moreover, Studies on both surgical biopsies and on lung cancer cells revealed that the expression level of ER beta and matrix-metalloproteinase-2 (MMP-2) were associated. Furthermore, inhibition of ER beta resulted in down-regulation of MMP-2 expression. Taken together, our results demonstrate that activation of ER beta in lung cancer cells promotes tumor metastasis through increasing expression of invasiveness-associated MMP-2. These results also highlight the therapeutic potential of inhibition of ER beta in the treatment of advanced NSCLC.