The 70-kDa heat shock protein (Hsp70) is up-regulated in a wide variety of tumor cell types and contributes to the resistance of these cells to the induction of cell death by anticancer drugs. Hsp70 binding protein 1 (HspBP1) modulates the activity of Hsp70 but its biological significance has remained unclear. We have now examined whether HspBP1 might interfere with the prosurvival function of Hsp70, which is mediated, at least in part, by inhibition of the death-associated permeabilization of lysosomal membranes. HspBP1 was found to be expressed at a higher level thanHsp70 in all normal and tumor cell types examined. Tumor cells with a high HspBP1/Hsp70 molar ratio were more susceptible to anticancer drugs thanwere those with a low ratio. Ectopic expression ofHspBP1 enhanced this effect of anticancer drugs in amanner that was both dependent on the ability of HspBP1 to bind to Hsp70 and sensitive to the induction of Hsp70 by mild heat shock. Furthermore, anticancer drugs upregulated HspBP1 expression, whereas prevention of such upregulation by RNA interference reduced the susceptibility of tumor cells to anticancer drugs.Overexpression ofHspBP1 promoted the permeabilization of lysosomal membranes, the release of cathepsins from lysosomes into the cytosol, and the activation of caspase-3 induced by anticancer drugs. These results suggest that HspBP1, by antagonizing the prosurvival activity of Hsp70, sensitizes tumor cells to cathepsin-mediated cell death.