The non-invasive imaging and radiotherapy by sodium/iodine symporter (NIS) gene transfer have been widely used for many experiments and some clinical studies. Baculovirus is an efficient tool for gene delivery into mammalian cells in vitro and in vivo. However, the applications of NIS and/or baculovirus in nasopharyngeal carcinoma (NPC) cells have not been reported yet. In this study, two recombinant baculoviruses expressing, respectively, NIS and green fluorescent protein (GFP), both under the control of the cytomegalovirus promoter (Bac-NIS and Bac-GFP) were successfully constructed. The infection efficiency and GFP fluorescence intensity of the human NPC cell line CNE-2Z infected by Bac-GFP at different setting of multiplicity of infection (MOI) were determined by flow cytometry. NIS protein expression was detected by indirect immunofluorescence. The 125I uptake and efflux of infected CNE-2Z cells by Bac-NIS were measured by a γ-counter. The cytotoxicity of baculovirus and sodium butyrate and inhibition of iodine uptake by NaClO4 were examined. The radioactivity and GFP fluorescence intensity in co-infected CNE-2Z cells by Bac-NIS and Bac-GFP were measured. Cell colony formation tests were conducted to evaluate the killing effect of Bac-NIS-mediated 131I. Based on the results, the transduction efficiency of Bac-GFP at the MOI of 200 or 400 reached 91.16 and 94.79%, respectively. NIS protein was expressed accurately on transfected CNE-2Z cell membranes and performed its normal function in iodine transport. Baculovirus had hardly any cytotoxic effects on infected cells, while relatively high concentration of sodium butyrate generated cytotoxicity. The correlation coefficient between the GFP fluorescence intensity and radioactivity in co-infected CNE-2Z cells was 0.917. Treatment coupled Bac-NIS with 131I killed the infected tumour cells dramatically in vitro. These results suggest that baculovirus is an effective vector of the gene delivery into CNE-2Z cells and NIS-mediated iodine transport is a potential approach for molecular imaging and radionuclide therapy of NPC.