Objective To investigate the chemosensitization of Chk2 inhibition to 5-FU against nasopharyngeal squamous epithelial cell line SUNE-1. Methods Chk2 siRNA was designed according to the mRNA sequence of Chk2, and its effect was confirmed by RT-PCR and Western blotting in SUNE-1 cells. Cell growth assay was evaluated to determine the impact of Chk2 siRNA on the proliferation ability of SUNE-1 cells and MTT assay was used to determine the chemosensitization of Chk2 siRNA to 5-FU. Finally, apoptotic cells was stained with Annexin Ⅴ/PI and examined by flow cytometry. Results Chk2 siRNA significantly reduced mRNA and protein levels of Chk2 in SUNE-1 cells, which confirmed the knockdown effect of Chk2 siRNA. Cell growth assay showed that Chk2 siRNA did not influence the proliferation of SUNE-1 cells. However, Chk2 knockdown significantly enhanced the cytotoxicity and apoptosis of SUNE-1 cells caused by 5-FU. Conclusion Chk2 hinders the toxic effect of 5-FU on SUNE-1 cells, which can be enhanced by Chk2 inhibition. Therefore, Chk2 can be proposed as a target to increase the effect of chemotherapeutic agent, such as 5-FU, in clinical treatment on nasopharyngeal carcinoma.