The phenolic phytoalexin resveratrol is well known for its health-promoting and anticancer properties. Its potential benefits are, however, limited due to its low bioavailability. Pterostilbene, a natural dimethoxylated analog of resveratrol, presents higher anticancer activity than resveratrol. The mechanisms by which this polyphenol acts against cancer cells are, however, unclear. Here, we show that pterostilbene effectively inhibits cancer cell growth and stimulates apoptosis and autophagosome accumulation in cancer cells of various origins. However, these mechanisms are not determinant in cell demise. Pterostilbene promotes cancer cell death via a mechanism involving lysosomal membrane permeabilization. Different grades of susceptibility were observed among the different cancer cells depending on their lysosomal heat shock protein 70 (HSP70) content, a known stabilizer of lysosomal membranes. A375 melanoma and A549 lung cancer cells with low levels of HSP70 showed high susceptibility to pterostilbene, whereas HT29 colon and MCF7 breast cancer cells with higher levels of HSP70 were more resistant. Inhibition of HSP70 expression increased susceptibility of HT29 colon and MCF7 breast cancer cells to pterostilbene. Our data indicate that lysosomal membrane permeabilization is the main cell death pathway triggered by pterostilbene.