The migration of different cell types, such as leucocytes and tumour cells, involves cellular strategies to overcome the physical resistance of three-dimensional tissue networks, including proteolytic degradation of extracellular matrix (ECM) components. High-resolution live-cell imaging techniques have recently provided structural and biochemical insight into the differential use of matrix-degrading enzymes in the migration processes of different cell types within the three-dimensional ECM. Proteolytic migration is achieved by slow-moving cells, such as fibroblasts and mesenchymally moving tumour cells, by engaging matrix metalloproteinases, cathepsins and serine proteases at the cell surface in a focalized manner ('pericellular proteolysis'), while adhesion and migratory traction are provided by integrins. Pericellular breakdown of ECM components generates localized matrix defects and remodelling along migration tracks. In contrast with tumour cells, constitutive non-proteolytic migration is used by rapidly moving T lymphocytes. This migration type does not generate proteolytic matrix remodelling, but rather depends on shape change to allow cells to glide and squeeze through gaps and trails present in connective tissues. In addition, constitutive proteolytic migration can be converted into non-proteolytic movement by protease inhibitors. After the simultaneous inhibition of matrix metalloproteinases, serine/threonine proteases and cysteine proteases in tumour cells undergoing proteolysis-dependent movement, a fundamental adaptation towards amoeboid movement is able to sustain non-proteolytic migration in these tumour cells (the mesenchymal-amoeboid transition). Instead of using proteases for matrix degradation, the tumour cells use leucoyte-like strategies of shape change and squeezing through matrix gaps along tissue scaffolds. The diversity of protease function in cell migration by different cell types highlights response diversity and molecular adaptation of cell migration upon pharmacotherapeutic protease inhibitor treatment.