While estrogen receptor β (ERβ) may impact the progression of non-small cell lung cancer (NSCLC), its linkage to alteration of the vasculogenic mimicry (VM) formation to influence the NSCLC cell invasion remains unclear. Here, we analyzed immunohistochemistry data from NSCLC tissues and found that ERβ-positive NSCLC female patients had worse survival outcomes than those of ERβ-negative NSCLC female patients. In vitro studies using multiple NSCLC cell lines also revealed that ERβ could increase the VM formation and cell invasion. Molecular mechanism dissection suggested that ERβ could increase the lncRNA-MALAT1 (MALAT1) expression via directly binding to the estrogen response elements (EREs) located on the promoter of MALAT1, which could then lead to (i) suppressing the miR145-5p and (ii) increasing the NEDD9 protein expression as miR145-5p can directly target the 3'-UTR of NEDD9-mRNA. A preclinical study using the in vivo mouse model further confirmed the in vitro cell lines data. Together, results from the above studies demonstrated that ERβ can promote NSCLC VM formation and cell invasion via altering the ERβ/MALAT1/miR145-5p/NEDD9 signaling. Targeting this newly identified signaling pathway with small molecules may help the development of novel therapies to better suppress the NSCLC metastasis.