Glioma-specific transcription of tumor-killing genes has been exploited as a promising gene therapeutic modality in glioma patients. Musashi1 (Msi1) and GFAP gene promoters are both cancer-specific promoters. Optimized HIF-binding site (optHBS) sequence was newly found as efficient as EPO HREs used as enhancer in cancer gene therapy. We constructed 4optHBS-Msi1/GFAP promoters and tested their ability to mediate BAX expression to induce apoptosis in glioma cell lines. Our results demonstrated that 4optHBS-Msi1/GFAP promoters are apparently strong and glioma-selective promoters with potential application in targeted glioma gene therapy, and 4optHBS-Msi1/GFAPBAXa are valuable tools for glioma gene therapy.