Abstract Background Normal epithelial cells and carcinoma cells can acquire invasiveness by epithelial-to-mesenchymal transition (EMT), a process of considerable cellular remodeling. The endosomal/lysosomal compartment is a principal site of intracellular protein degradation. Lysosomal cathepsin proteases are secreted during cancer progression. The established pro-metastatic role of specific cysteine cathepsins has until now been ascribed to their contribution to extracellular matrix remodeling. We hypothesized that cysteine cathepsins affect transforming growth factor β-1 (TGFβ-1)-induced EMT of normal and malignant mammary epithelial cells. Methods The role of lysosomal proteolysis in TGFβ-1-induced EMT and invasion was investigated in a normal and a novel malignant murine mammary epithelial cell line. The contribution of cysteine cathepsins was determined by addition of the general cysteine cathepsin inhibitor E64d. Hallmarks of EMT were analyzed by molecular- and cell-biologic analyses including real-time cell migration/invasion assays. A quantitative proteome comparison using stable isotopic labeling with amino acids in culture (SILAC) showed the effect of E64d on TGFβ-1 induced proteome changes. Lysosomal patterning and junctional adhesion molecule A (Jam-a) localization and abundance were analyzed by immunofluorescence. Results We found increased lysosome activity during EMT of malignant mammary epithelial cells. Cysteine cathepsin inhibition had no effect on the induction of the TGFβ-1-induced EMT program on transcriptional level. Protease inhibition did not affect invasion of TGFβ-1 treated normal mammary epithelial cells, but reduced the invasion of murine breast cancer cells. Remarkably, reduced invasion was also evident if E64d was removed 24 h before the invasion assay in order to allow for recovery of cathepsin activity. Proteome analyses revealed a high abundance of lysosomal enzymes and lysosome-associated proteins in cancer cells treated with TGFβ-1 and E64d. An accumulation of those proteins and of lysosomal vesicles was further confirmed by independent methods. Interestingly, E64d caused lysosomal accumulation of Jam-a, a tight junction component facilitating epithelial cell-cell adhesion. Conclusion Our results demonstrate an important role of lysosomal proteolysis in cellular remodeling during EMT and a pivotal contribution of lysosomal cysteine cathepsins to TGFβ-1 induced acquisition of breast cancer cell invasiveness. These findings provide an .