B-cell lymphoma-extra large (Bcl-xL), an important member of anti-apoptotic Bcl-2 family, is involved in tumor progression and development. The overexpression of Bcl-xL is associated with radioresistance of human malignancies. The present study aimed to investigate the inhibitory effect of small interfering RNA (siRNA) on the expression of Bcl-xL in the A549 non-small lung cancer (NSCLC) cell line, and its role in inducing the apoptosis and increasing the radiosensitivity of A549 cells. An siRNA expression vector, pSilencer4-CMVneo-short hairpin (sh)RNA, was constructed and stably transfected into A549 cells. The effects of Bcl-xL-shRNA on cell proliferation, apoptosis and the protein expression levels of associated proteins were assessed in vitro in the A549 cells. The radiosensitivity of the A549 cells was evaluated using a clonogenic cell survival assay. The results demonstrated that the sequence-specific siRNA targeting Bcl-xL efficiently and specifically downregulated the mRNA and protein expression levels of Bcl-xL. The RNA interference-mediated downregulation in the expression of Bcl-xL inhibited cell proliferation, induced apoptosis and reduced the radioresistance of the NSCLC cells. These findings suggested that Bcl-xL may be a promising therapeutic approach for the treatment of NSCLC.