Lung cancer is reported to be a major public health issue worldwide and the overall prognosis of patients remains poor. The expression levels of Livin and Survivin, of the inhibitors of apoptosis (IAP) family, are associated with prognostic significance in the majority of solid tumors. Therefore, in the presents study, short hairpin (sh)RNA expression vectors inhibiting the Livin and Survivin genes were constructed to examine the effects of the transfection of Livin shRNA and/or Survivin shRNA on the biological functions of tumor cells. The transfection efficiency was measured using fluorescence reverse transcription‑quantitative polymerase chain reaction and western blot analyses. The cell growth inhibition ratio was measured using a CCK assay. Cell apoptosis following transfection and in tumor tissues were measured using a TUNEL assay, and a cancer xenograft model was used to investigate the effect of Livin shRNA and/or Survivin shRNA on tumor growth. The results indicated that the mRNA and protein expression levels were suppressed following the transfection of Livin and Survivin shRNA into tumor cells (P<0.05, compared with control group). The growth of tumor cells in vivo and in vitro was significantly inhibited following transfection with Livin and Survivin shRNA, compared with that in the other groups (P<0.05). Taken together, the transfection of cells with Livin and Survivin inhibited tumor growth in vivo and in vitro, with the co‑transfection of Livin and Survivin shRNA showing increased efficiency, compared with transfection of either the Livin vector or Survivin vector alone. The combined inhibition of Livin and Survivin may be a promising multitargeted gene therapeutic strategy in cancer treatment.