Chaetocin, a fungal metabolite, exerts notable antiproliferative effects against solid tumors by triggering apoptosis; however, the mechanisms underlying its effects remain unclear. As tumor necrosis factor (TNF)‑related apoptosis‑inducing ligand (TRAIL) promotes apoptosis in certain types of tumor, the present study aimed to explore the sensitizing effects of chaetocin in TRAIL‑induced apoptosis in human glioblastoma cells and the underlying mechanism. Human glioblastoma cells (U343MG, U87MG, U251MG, and T98G) and embryonic kidney cells (HEK293) were co‑treated with chaetocin and TRAIL, followed by assessment of cell viability. The results from viability and apoptosis assays demonstrated a significant increase in caspase-dependent apoptosis in glioblastoma cells, but not in HEK293 cells, upon co-treatment with chaetocin and TRAIL. Additionally, death receptor 5 (DR5) expression analysis demonstrated that co‑treatment with chaetocin and TRAIL upregulated DR5 expression in a dose‑ and time‑dependent manner by increasing the stability of DR5 on the cell surface. In glioblastoma cells, small interfering RNA‑mediated DR5 knockdown markedly suppressed chaetocin/TRAIL‑induced apoptosis. Moreover, chaetocin enhanced reactive oxygen species (ROS) production, which facilitated TRAIL‑mediated apoptosis by enhancing DR5 upregulation. Thus, chaetocin sensitized the human glioblastoma cell lines U87MG and T98G to TRAIL‑mediated apoptosis by upregulating DR5 expression through ROS-mediated mechanisms. The present findings underscore chaetocin as a potential novel therapeutic agent for glioblastoma.