Objective： To construct a replicating adenovirus vector CNHK600-p53 carrying p53 gene and investigate its effect on the chemosensitivity of hepatocarcinoma cell line. Methods： Chemotherapy of hepatocarcinoma cells BEL-7404 were carried out using Fluorouracil, Mitomycin, Epirubicin, CNHK600-p53, CNHK600-p53 ＋ Fluorouracil, CNHK600-p53 ＋ Mitomycin, and CNHK600-p53 ＋ Epirubicin, separately. MTT assay was used to evaluate the killing effects after therapy. Resuits： The inhibition rate on BEL-7404 cells was（47±3）% when using 100 μg/ml 5-Fu, was （20±4）% when using 1 μg/ml MMC, and was （73±2）% when using 2.5 μg/ml EPI. The inhibition rates of BEL-740 cells in CNHK600-p53（MOI= 10） ＋ Fluorouracil （100 μg/ml）, CNHK600-p53（MOI=10） ＋ Mitomycin （1 μg/ml）, and CNHK600-p53 （MOI= 10）＋ Epirubicin （2.5 μg/ml） groups were （59±4） %, （44±4） % and （86±2） %, respectively （P〈0.01）. Conclusion： Adenovirus CNHK600- p53 carrying p53 gene can enhance chemosensitivity of BELT404 hepatocarcinoma cell line. Gene therapy using adenovirus CNHK600-p53 in combination with chemotherapy may be a new strategy for hepatocarcinoma treatment.