HIV type 1 (HIV-1) protease inhibitors (PI) have been shown to have anticancer activity in non-HIV-associated human cancer cells. The underlying mechanism of this effect is unclear. Here, we show that the PIs nelfinavir and atazanavir cause cell death in various malignant glioma cell lines in vitro. The underlying mechanism of this antitumor effect involves the potent stimulation of the endoplasmic reticulum (ER) stress response (ESR), as indicated by increased expression of two ESR markers, GRP78 and CHOP, and activation of ESR-associated caspase-4. Induction of ESR seems to play a central role in PI-induced cell death because small interfering RNA-mediated knockdown of the protective ER chaperone GRP78 sensitizes cells; whereas knockdown of proapoptotic caspase-4 protects cells from PI-induced cell death. Furthermore, the treatment of cells with PIs leads to aggresome formation and accumulation of polyubiquitinated proteins, implying proteasome inhibition. Thus, our results support a model whereby PIs cause tumor cell death via triggering of the ESR, inhibition of proteasome activity, and subsequent accumulation of misfolded proteins. Inhibition of glioma growth via ESR takes place in the in vivo setting as well, as nelfinavir inhibits the growth of xenografted human malignant glioma, with concomitant induction of the proapoptotic ER stress marker CHOP. Because ER stress has also been reported as the mechanism for insulin resistance and diabetes, our ER stress model of PI function may also explain why these drugs may induce insulin resistance as one of their most common side effects.