Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine with potential anticancer effect, but innate and adaptive TRAIL resistance in majority of cancers limit its clinical application. Karyopherin β1 (KPNB1) inhibition in cancer cells has been reported to abrogate the nuclear import of TRAIL receptor DR5 and facilitate its localization on the cell surface ready for TRAIL stimulation. However, our study reveals a more complicated mechanism. Genetic or pharmacological inhibition of KPNB1 potentiated TRAIL-induced apoptosis selectively in glioblastoma cells mainly by unfolded protein response (UPR). First, it augmented ATF4-mediated DR5 expression and promoted the assembly of death-inducing signaling complex (DISC). Second, it freed Bax and Bak from Mcl-1. Third, it downregulated FLIPL and FLIPS, inhibitors of caspase-8 cleavage, partly through upregulating ATF4-induced 4E-BP1 expression and disrupting the cap-dependent translation initiation. Meanwhile, KPNB1 inhibition-induced undesirable autophagy and accelerated cleaved caspase-8 clearance. Inhibition of autophagic flux maintained cleaved caspase-8 and aggravated apoptosis induced by KPNB1 inhibitor plus TRAIL, which were abolished by caspase-8 inhibitor. These results unveil new molecular mechanism for optimizing TRAIL-directed therapeutic efficacy against cancer.