RALY is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP), an RNA-binding protein that plays a role in mRNA splicing and metabolism, may be involved in tumorigenesis and development. Some studies have shown that RALY plays a role in promoting cancer in a variety of tumors. However, the biological function and molecular mechanism of RALY in non-small cell lung cancer (NSCLC) remain unknown. TCGA databases were used to gather RALY expression data in NSCLC, the results indicate that RALY is highly expressed in cancer tissue of NSCLC patients. Then we demonstrated that RALY gene expression was notably upregulated in NSCLC tissue and cell lines (A549 and SK-MES-1), and was associated with lymph node metastasis (P = 0.007) and poorer overall survival in NSCLC patients. Subsequently, RALY in A549 and SK-MES-1 cells was knocked down by lentivirus to analyze the consequences of RALY on the biological behavior of NSCLC cell lines. Our results indicated that RALY knockdown impaired NSCLC cells proliferation, migration, and invasion, as well as arrested cells in G1 phase, and the reintroduction of RALY recused its biological phenotype. Furthermore, RALY knockdown down-regulated the expression levels of c-Myc, Cyclin D1, CDK4, MMP9, Rho A ,Rho C, N-cadherin and β-catenin, and up-regulated the expression levels of P27, Rho B and E-cadherin. Therefore, targeting RALY could be a promising molecular target for NSCLC treatment.