Glioblastoma multiforme (GBM) is by far the most common and most malignant of primary brain tumors. Total surgical resection is not possible, due to diffuse single cell infiltration into surrounding healthy brain parenchyma. Additionaly, heterogeneity of tumor and heterogeneity of GBM cells itself makes GBM resistant to conventional cancer therapy. Consequently, GBM relapse is inevitable and patients die within 15 months after being diagnosed. Mesenchymal stem cells of bone marrow origin (MSC) exert tropism to GBM, and have the ability to modulate immune system and form functional and structural syncytium with GBM cells within GBM microenvironment. That is why MSC represent an interesting new transport vehicle for drug delivery into the tumor. The aim of this research was to evaluate the effect of co-culturing of MSC and U373 GBM cells on protease expression of calpain 1, calpain 2, cathepsin B, cathepsin K, cathepsin L in both cell types. We proved that the expression of the selected proteases was higher in MSC than in U373 cells, and that calpain 1 and cathepsin B were up-regulated in U373 cells when they were grown in the co-culture with MSC. In contrast, the expression of calpain 1 and cathepsin B was decreased in MSC, wich were co-cultured with U373 cells. Based on these results, we conclude that the cells in the tumor microenvironment significantly influence each other and cell invasiveness is changed in co-cultures, when compared to monocultures. Aditional research on interactions between stromal cells, such as MSC, and GBM cells is needed to aquire a more precise information about behaviour of individual cell type in the tumor microenvironment. The knowledge of proteases in tumour microenvironment can lead to an advanced cell therapy of GBM.