The multiple drug resistance protein (MDR1) is frequently overexpressed in human glioma. The aim of this study is to clone the MDR1 promoter from C6/ADR, construct the double suicide genes expressive vector controlled by MDR1 promoter, and explore its targeted expression in C6/ADR cells. MDR1 promoter from C6/ADR genomic DNA, which was linked with T vector, was amplified by using Polymerase chain reaction (PCR). After cut by NdeI and HindIII, MDR1 promoter was cloned into pcDNA3-TK (thymidine kinase) plasmid. The cytosine deaminase (CD) gene from pcDNA3-CD-TK plasmid was directly cloned into the above vector to construct pcDNA3-MDR1-promoter-CD-TK vector. Then this vector was transfected into C6 and C6/ADR cells respectively by liposome. After selection by G418, the tumor cell lines were stably established. Then these cell lines were examined through PCR and RT-PCR to respectively detect the integration and expression of TK and CD genes. The results showed the length and sequence of MDR1 promoter amplified by PCR were confirmed by DNA sequencing. The pcDNA3-MDR1-promoter-CD-TK expression vectors were constructed successfully. PCR indicated the double suicide genes were integrated into C6 and C6/ADR cells. RT-PCR revealed that CD and TK genes expressed in C6/ADR/CD-TK cells, whereas not in C6/CD-TK cells. In conclusions, construction of expressive vector containing double suicide genes controlled by MDR1 promoter with targeted expression in C6/ADR will provide a sound basis for targeted gene therapy for multidrug resistance (MDR) glioma.