Objective To investigate the feasibility of baculovirus-mediated ¹³¹I radiotherapy by carrying sodium iodide symporter (NIS) gene for hepatocellular carcinoma. Methods The recombinant baculoviruses carrying green fluorescent protein （GFP）or NIS gene (Bac-GFP and Bac-NIS) were prepared by Bac-to-Bac baculovirus expression system. The infection efficiency and fluorescence intensity of HepG2 cells were determined by flow cytometry in the presence or absence of sodium butyrate, and the cytotoxic effect of baculovirus on HepG2 cells was also determined in the condition of high multiplicity of infection (MOI). Furthermore, HepG2 cells infected with Bac-NIS were examined with dynamic iodide uptake test and natrium perchloricum (NaClO4) iodide uptake inhibition test, and cell clone formation assay was adopted to evaluate the killing efficacy of ¹³¹I in HepG2 cells infected with Bac-NIS. Results The infection efficiency and fluorescence intensity of HepG2 cells increased along with the increase of Bac-GFP MOI. The infection efficiency of Bac-GFP was significantly higher in HepG2 cells (MOI=200, infection efficiency was 65%), and sodium butyrate could improve the infection efficiency of HepG2 cells. In the condition of high MOI (MOI=400), there was no cytotoxicity induced by Bac-GFP on HepG2 cells, and was not significantly different from that of control group (MOI=0)(P＞0.05). HepG2 cells infected with Bac-NIS exhibited functional iodide uptake and the property of NaClO4 inhibition. Cell clone formation assay revealed that the clone formation rate of HepG2 cells infected with Bac-NIS was significantly lower than that of HepG2 cells infected with Bac-GFP （P＜0.01）. Conclusion Bac-NIS can effectively mediate iodine uptake and has high killing effects on hepatic carcinoma cells, which may provide experimental basis for cell targeted gene radiotherapy for hepatocellular carcinoma.