Signal transducer and activator of transcription 3 (STAT3) is an oncogene aberrantly activated in many human tumors. We studied whether radiation combined with STAT3 siRNA enhances radiosensitivity of Hep-2 human laryngeal squamous carcinoma cells (Hep-2 cells). Firstly, STAT3 targeting recombinant plasmid was constructed. Hep-2 cells were transfected with expression vector of STAT3 siRNA using Lipofectamine 2000. Semiquantitive RT-PCR detected effective STAT3 mRNA down-regulation by STAT3 siRNA. Secondly, Hep-2 cells were radiated with different doses of gamma-rays after transfection with STAT3 small interference RNA (siRNA). MTT assay showed cell proliferation decreased significantly (P<0.05) after STAT3 siRNA transfection combined with radiation. Thirdly, flow cytometry (FCM) demonstrated that cell apoptosis of combined treatment group increased significantly (P<0.05) and exhibited time dependency after 6 Gy irradiation (P<0.05). Simultaneously, STAT3, p-STAT3, Bcl-2, VEGF, p53 protein levels decreased in Hep-2 cells, with positive correlations between level of p-STAT3 and levels of Bcl-2, VEGF, p53, respectively (r=0.974, 0.988, 0.976, all P<0.01). Above all, specific siRNA targeting STAT3 gene is able to enhance the radiosensitivity in Hep-2 cells by regulating expression of Bcl-2, VEGF and p53 proteins.