MT-SP1 is a type II transmembrane serine protease implicated in a range of human cancers including those of the breast, cervix, ovaries, prostate, colon and gastrointestinal tract. Mouse models have shown it to be critical for proper epidermal development and postnatal survival. However, the role of this enzyme in normal and malignant biology has not yet been fully elucidated. Several groups have identified putative substrates of MT-SP1 in an effort to understand the possible biological processes in which this protease may be involved. Methods for substrate identification include comparing known protein cleavage sequences with MT-SP1 specificity data, in vitro cleavage assays, examining genetic microarrays for enzyme/substrate coexpression, immunohistochemistry for colocalization, and a variety of phenotypic observations using cell culture and mouse models. Given the inherent limitations of each individual method, substrate plausibility is best substantiated using a combination of experimental approaches. Here we review MT-SP1 substrates identified to date and the possible physiological implications of substrate cleavage in cell-microenvironment interactions. This data indicates that MT-SP1 is capable of playing roles in growth factor activation, receptor activation and inactivation, protease activation, and ectodomain shedding. We also present for the first time vascular endothelial growth factor receptor 2 (VEGFR-2) as a putative substrate for MT-SP1.