Increases in protease expression and activity are associated with malignant progression and poor patient prognosis in a number of human cancers. Members of the papain family of cysteine cathepsins are among the protease classes that have been functionally implicated in cancer. Inhibition of the cysteine cathepsin family using a pan-cathepsin inhibitor, JPM-OEt, led to tumor regression in the RIP1-Tag2 (RT2) mouse model of pancreatic islet cell tumorigenesis. The present study was designed to determine whether this cathepsin inhibitor, when used in combination with chemotherapy, would increase antitumor efficacy. RT2 mice were treated in a late-stage regression trial with three different chemotherapy regimens, alone or in combination with the cathepsin inhibitor, JPM-OEt. Cyclophosphamide was administered in either a maximum tolerated dose (MTD) regimen, a “metronomic” continuous low-dose regimen, or a “chemo-switch” regimen consisting of MTD followed by metronomic dosing. Mice were sacrificed at a defined end point and tumor burden was assessed followed by a detailed analysis of cell proliferation, apoptosis, vascularization, and invasiveness in the treated and control lesions. An additional cohort of mice was followed for survival analysis. The cathepsin inhibitor plus the chemo-switch regimen of cyclophosphamide led to the most pronounced reduction in tumor burden and greatest increase in overall survival. Cysteine cathepsin inhibition resulted in a significant decrease in tumor invasiveness, which was further augmented in combination with each of the chemotherapy dosing regimens. These results encourage the development and continuing evaluation of cysteine cathepsin inhibitors as cancer therapeutics. [Cancer Res 2007;67(15):7378–85]