The therapeutic options available for the treatment of advanced non-small cell lung cancer have increased over the past decade. Small molecule gene therapy has emerged as an effective therapy for the treatment of lung cancer in vitro and in vivo although it has not been tested in a clinical setting. In particular, therapies that target the negative feedback loop between p53 and murine double minute 2 (MDM2) provide a favorable outcome by maintaining activation of the tumor suppressor gene p53. The present study used transfection to simultaneously knockdown MDM2 expression using small interfering (si)RNA, and overexpress wild‑type p53 in H1299 cells. The effects of transfection on cell proliferation and cell cycle progression were determined using an MTT assay and flow cytometry, and the effects on mRNA and protein expression were determined by western blotting and reverse transcription polymerase chain reaction. The results indicated that simultaneously knocking down MDM2 and overexpressing p53 was able to inhibit proliferation and induce G1 cell cycle arrest in H1299 cells, compared with either alone. These findings indicated that the si‑MDM2‑p53 co‑expression plasmid may induce cell cycle arrest, and may be considered a novel therapeutic option for the treatment of lung cancer.