Objective To explore the effect of interfering RNA (shRNA) on biological activity of A549 cells and tumor growth in nude mice after knockdown of estrogen receptor α (ERα) gene. Methods The ERα gene in A549 cells was knocked down by shRNA. RT-PCR and Western blot were used to detect the gene expression and protein expression after knockdown; colony formation experiment was used to detect the proliferation of cells, and RT-PCR was used to detect the expression of Ki-67 and PCNA; flow cytometry was used to detect apoptosis rate; transwell assay was used to detect cell invasion ability; Western blot was used to detect the expression of epithelial cadherin (E-cad) and neuropathic cadherin (N-cad) protein. The control group and A549 cells transfected with ERα-shRNA1 were injected subcutaneously in nude mice to construct transplanted tumors. Immunohistochemistry was used to detect the expression of Ki-67 and N-cad in tumor tissues. Results Compared with the control group, after transfection of ERα-shRNA1 and ERα-shRNA2, the mRNA and protein expressions of ERα were reduced significantly (P<0.05), and shRNA1 with high interference efficiency was used for subsequent experiments. Compared with the control group, the A549 cells were transfected with ERα-shRNA1, the colony formation rate was down-regulated significantly (P<0.05), the apoptosis rate was increased significantly (P<0.05), the expression of Ki-67 and PCNA were down-regulated significantly (P<0.05), the number of invasive cells was reduced significantly, the expression of E-cad was increased, and the expression of N-cad was decreased (P<0.05). The results of tumor formation in nude mice showed that interfering with ERα expression can significantly inhibit tumor growth (P<0.05), and down-regulate the rate of Ki-67 and N-cad positive cells (P<0.05). Conclusion Knockdown of ERα inhibits the proliferation and migration ability of NSCLC cells and the occurrence and development of transplanted tumors in nude mice.