The invasion of glioblastoma is a complex process that involves coordinated interactions between tumor cells and the extracellular matrix. Biomaterial scaffolds now show promise in forming in vitro and in vivo three-dimensional (3D) cancer models and can generate an in vitro micro-environmental context that mimics the complexity of the tumor tissue in vivo. Higher expression of MMP-2 and MMP-9 and plasminogen activator in tumor cells is correlated with an increased grade of glioblastoma malignancy. In this study, we fabricated a microtumor and investigated the effect of viscosity of collagen hydrogel and the inhibitors of MMPs and plasminogen activators on migration of glioblastoma cells in collagen hydrogel. The cell viability and proliferation of microtumors were studied by LIVE/DEAD assay and AlamarBlue assay respectively. qRT-PCR was performed to study MMP-2, MMP-9 and plasminogen activator gene expression in collagen hydrogels. Time-lapse imaging was recorded to quantify tumor cell migration speed and distance in collagen hydrogel and in collagen hydrogels containing inhibitors of MMP-2, MMP-9 and plasminogen activator. Crosslinking of collagen hydrogels increased the viscosity of collagen hydrogel and decreased tumor cell migration distance and velocity. Crosslinking reduced the tumor cell-mediated degradation rate of the hydrogels. The inhibitors of MMP-2 and MMP-9 reduced tumor cell migration speed on cell culture dishes or in hydrogels. Although tranexamic acid and aprotinin did not change the tumor cell migration on cell culture dish, they significantly reduced the cell migration in collagen hydrogels. The similar inhibition effect of MMP-2 and MMP-9 inhibitors on tumor cell migration on cell culture dish and in hydrogels was observed in this study. This study demonstrated that the increased ECM viscosity can limit glioma cell migration and demonstrates the different functions of pharmacologic inhibitors on glioma cell motility in a 3D matrix against a 2D environment.