Glioblastoma is a highly aggressive and malignant type of cancer that is apoptosis resistant and difficult to cure by conventional cancer therapies. In this regard, an oncolytic adenovirus that selectively targets the tumour tissue and induces tumour cell lysis is a promising treatment option. We designed and constructed a hypoxia-responsive and cancer-specific modified human telomerase reverse transcriptase (H5CmTERT) promoter to drive replication of an oncolytic adenovirus (H5CmTERT-Ad). To enhance the anti-tumour efficacy of H5CmTERT-Ad against malignant glioblastoma, we also generated an H5CmTERT-Ad expressing secretable trimeric tumour necrosis factor-related apoptosis-inducing ligand (H5CmTERT-Ad/TRAIL). H5CmTERT promoter-regulated oncolytic adenoviruses showed cancer-specific and superior cell-killing effect in contrast to a cognate control oncolytic adenovirus replicating under the control of the endogenous adenovirus promoter. The cancer cell-killing effects of H5CmTERT-Ad and H5CmTERT-Ad/TRAIL were markedly higher during hypoxia than normoxia owing to hypoxia responsiveness of the promoter. H5CmTERT-Ad/TRAIL showed more potent anti-tumour efficacy than H5CmTERT-Ad did in a xenograft model of TRAIL-resistant subcutaneous and orthotopic glioblastoma through superior induction of apoptosis and more extensive virus distribution in the tumour tissue. Altogether, our findings show that H5CmTERT-Ad/TRAIL can promote dispersion of an oncolytic adenovirus through robust induction of apoptosis in a highly TRAIL-resistant glioblastoma.