Objective To investigate the killing effect of herpes simplex virus thymidine kinase/gancyclovir (HSV-tk/GCV) suicide gene therapy system on retinoblastoma (Rb) cells and the mechanism of bystander effect. Methods By using liposome, pCMV/hytk-IREShrGFP plasmid was transferred into HXO-Rb44 cells. A fluorescence microscope was used to detect the transduction effeciency. The positive cell clones were selected by hygromycin and were named HXO-Rb44/tk. RT-PCR was resorted to demonstrate the sucessful transduction and transcription of hytk gene in the HXO-Rb44/tk cells. The morphologic features and growth patterns of HXO-Rb44/tk were compared with those of HXO-Rb44. Then MTT assay was used to determined the killing effect of GCV on HXO-Rb44/tk and the mixture of HXO-Rb44/tk and HXO-Rb44 in different ratios ("bystander effect"). The mechanism of bystander effect was studied by the experiment of supernatant shifting. Results The transduction effeciency was 20%. 530bp hytk gene strand was seen through HXO-Rb44/tk RT-PCR. There were no differences in the morphologic features or the growth patterns between HXO-Rb44/tk and HXO-Rb44. HXO-Rb44/tk was more sensitive to GCV than was HXO-Rb44. The cytotoxicity of HXO-Rb44/tk was dose and time dependent. An obvious "bystander effect" was seen even with low proportions of HXO-Rb44/tk, but this effect disappeared when transferring GCV containing supernatant of HSV-tk-positive cells to the negative cells. Conclusion The transfer of the HSV-tk gene into Rb cells followed by the administration of GCV could serve as a model for gene therapy for retinoblastoma, the "bystander effect" in HSV-tk/GCV-mediated gene therapy occurs by transfer of GCV metabolite from cell to cell through gap junction.