Temozolomide (TMZ) is widely used for treating glioblastoma (GBM), which can effectively inhibit the GBM growth for some months; however, it still could not prevent the invariable recurrence of GBM. The existence of glioma stem cells (GSCs) was considered to be a key factor. But TMZ has poor effects on GSCs. Recently, demethoxycurcumin (DMC) has been shown to display anti-tumor activities in malignant gliomas. However, its effects and the potential mechanisms on GSCs were still unclear. Our study showed that DMC was prior to TMZ on resulting in a significant increase in GSC apoptosis and a marked inhibition of cell growth in vitro. And combined treatment of DMC and TMZ showed more significant anti-GSC effects. Further research into the underlying mechanism demonstrated that this novel combinatorial regimen leads to changes of multiple cell signaling pathways including reactive oxygen species (ROS) production and caspase-3 signaling mitochondria-related apoptosis activation as well as inactivation of JAK/STAT3 signaling pathway. Taken together, our data demonstrate that the anti-GSC effects of DMC are better than TMZ, and combined treatment of DMC and TMZ has much stronger effects on GSCs.