Cathepsin L is a lysosomal enzyme thought to play a key role in malignant transformation. Recent work from our laboratory has demonstrated that this enzyme may also regulate cancer cell resistance to chemotherapy. The present study was undertaken in order to define the relevance of targeting cathepsin L in suppression of drug resistance in vitro and in vivo, and also to understand the mechanism(s) of its action. In vitro experiments indicated that cancer cell adaptation to increased amounts of doxorubicin over time was prevented in the presence of cathepsin L inhibitor, suggesting that inhibition of this enzyme not only reverses, but also prevents the development of drug resistance. The combination of cathepsin L inhibitor with doxorubicin also strongly suppressed proliferation of drug resistant tumors in nude mice. Investigation of the underlying mechanism(s) led to the finding that the active form of this enzyme shuttles between the cytoplasm and nucleus. As a result, its inhibition stabilizes and enhances the availability of cytoplasmic and nuclear protein drug targets including the estrogen receptor , Bcr-Abl, the topoisomerase-II, the histone deacetylase 1, and the androgen receptor. In support of this, cellular response to doxorubicin, tamoxifen, Imatinib, Trichostatin A and flutamide increased in the presence of cathepsin L inhibitor. Together, these findings provided evidence for the potential role of cathepsin L as a target to suppress cancer resistance to chemotherapy, and uncovered a novel mechanism by which protease inhibition-mediated drug target stabilization may enhance cellular visibility and thus, susceptibility to anti-cancer agents.