Neuroblastoma arises as a direct result of genetic disorder; therefore, it should be well treated and conquered by gene therapy in future. In this study, neuroblastoma cell line SH-SY5Y experiments, in vitro and in nude mice in vivo, were subjected to research thymidine kinase suicide gene to treat neuroblastoma. The plasmid LXpsp-hytk and a plasmid LXSH were transduced separately by lipofectin into human neuroblastoma cell line SH-SY5Y. SH-SY5Y-hy and SH-SY5Y-hytk were selected by hygromycin B. Different ganciclovir (GCV) concentration was given to SH-SY5Y-hytk to determine optimal GCV concentration. The cytotoxic effect of GCV on SH-SY5Y-hytk, SH-SY5Y-hy, and SH-SY5Y cells was determined. Scapular subcutaneous tumors were established in nude mice by inoculating 2.5x10(6) SH-SY5Y-hytk on their left sides and 2.5x10(6) SH-SY5Y-hy cells on their right sides for every mouse of treatment group and control group, respectively. After 1 week, mass grew in both sides of all the mice, and from the eighth day on, every mouse in treatment group received daily intraperitoneal injection of GCV 50 mg/kg body weight for 14 days; every mouse in control group received daily intraperitoneal injection of 1 ml saline for 14 days. On day 22 tumors were excised and weighed on the left and right sides, respectively, and apoptosis was detected by TUNEL method. Apoptotic index was calculated on the left and on the right sides, respectively, for every mouse in treatment group and control group. The lowest concentration of hygromycin B was 60 mug/ml. The cytotoxic effect of GCV on SH-SY5Y-hytk cells was obvious (IC50=0.03 muM), whereas GCV showed almost no cytotoxic effect on SH-SY5Y and SH-SY5Y-hy cells (IC50>400 muM). SH-SY5Y-hytk was killed by concentrations of 30 muM GCV effectively and it obviously showed the bystander effect, when SH-SY5Y-hytk remained at least 18% in the mixture culture cells. The tumor on the left side was much smaller than that of the right side in control group (p<0.05), and apoptotic index of the left was higher than that of the right in control group (p<0.01). SH-SY5Y-hytk has the bystander effect over 18% SH-SY5Y-hytk of the mixture culture cells at the concentration of 30 muM GCV. The HSV-tk/GCV system was effective in treating SH-SY5Y neuroblastoma cell line in vivo as well. Our findings suggest that thymidine kinase gene therapy could be a potential method for treating neuroblastoma in the future.