A suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) with ganciclovir (GCV) has been under development as a tumor-targeted therapy; however, the mechanism of cellular GCV uptake, which is prerequisite in the therapy, has not been clarified. In an attempt to resolve this situation and gain information to optimize HSV-TK/GCV system for cancer therapy, we found that human equilibrative nucleobase transporter 1 (ENBT1) can transport GCV with a Michaelis constant of 2.75 mM in Madin-Darby canine kidney II (MDCKII) cells stably transfected with this transporter. In subsequent experiments using green fluorescent protein (GFP)-tagged ENBT1 (GFP-ENBT1) and HSV-TK, the uptake of GCV (30 μM), which was minimal in MDCKII cells and unchanged by their transfection with HSV-TK alone, was increased extensively by their transfection with GFP-ENBT1, together with HSV-TK. Accordingly, cytotoxicity, which was assessed by the WST-8 cell viability assay after the treatment of those cells with GCV (30 μM) for 72 hours, was induced in those transfected with GFP-ENBT1, together with HSV-TK but not in those transfected with HSV-TK alone. These results suggest that ENBT1 could facilitate GCV uptake and thereby enhance cytotoxicity in HSV-TK/GCV system. We also identified Helacyton gartleri (HeLa) and HepG2 as cancer cell lines that are rich with ENBT1 and A549, HCT-15 and MCF-7 as those poor with ENBT1. Accordingly, the HSV-TK/GCV system was effective in inducing cytotoxicity in the former but not in the latter. Thus, ENBT1 was found to be a GCV transporter that could enhance the performance of HSV-TK/GCV suicide gene therapy.