Background: To explore the impact of pU6-based tandem survivin and CDK1-specific short hairpin RNA on the biological behaviors of CNE-2 nasopharyngeal carcinoma cells in vitro and in vivo. Patients and Methods: The vectors of pU6-survivinshRNA, pU6-CDK1shRNA and pU6-survivinshRNA-CDK1shRNA were constructed and transfected into CNE-2 cells with Lipofectamine TM 2000, respectively. The mRNAs and proteins of CDK1 and survivin were determined by RT-PCR and Western blotting, accordingly. MTT assay was employed to evaluate the proliferation of CNE-2 cells, and flow cytometry was performed to determine the apoptosis of CNE-2 cells. The effects of interfering survivin and CDK1 on tumorigenesis were evaluated by tumor xenografts experiments. Results: Effective plasmids were successfully constructed knocking down survivin and/or CDK1. The proliferation inhibition of CNE-2 cells by pU6-survivinshRNA-CDK1shRNA (32.5%) was higher than that of by pU6-survivinshRNA (25.6%) and pU6-CDK1shRNA (15.6%), and apoptosis in CNE-2 cells simultaneously interfering survivin and CDK1 (15.2%) dramatically increased when compared to those of interfering survivin (5.4%) or CDK1 (4.7%) alone. Furthermore, simultaneously interfering survivin and CDK1 is more effective than interfering alone component in inhibiting tumor growth of fBalb/C nude mice xenografted with CNE-2 cells. Conclusion: The results altogether indicate that interfering survivin and CDK1simutaneously can produce synergistic effects of anti-nasopharyngeal carcinoma, which could be a potential therapeutic method.