The urokinase-type plasminogen activator receptor (uPAR) has been implicated in several processes in tumor progression including cell migration and invasion in addition to initiation of signal transduction. Since uPAR lacks a transmembrane domain, it uses the interaction with other proteins to modulate intracellular signal transduction. We have previously identified hSpry1 as a partner protein of uPAR, suggesting a physiological role for hSpry1 in the regulation of uPAR signal transduction. In this study, hSpry1 was found to colocalize with uPAR upon stimulation with epidermal growth factor (EGF), urokinase (uPA), or its amino terminal fragment (uPA-ATF), implicating a physiological role of hSpry1 in regulation of uPAR signalling pathway. Moreover, hSpry1 was able to inhibit uPAR-stimulated cell migration in HEK293/uPAR, breast carcinoma, and colorectal carcinoma cells. In addition, hSpry1 was found to inhibit uPAR-stimulated cell invasion in breast carcinoma and osteosarcoma cell lines. Increasing our understanding of how hSpry1 negatively regulates uPAR-stimulated cellular functions may determine a distinctive role for hSpry1 in tumour suppression.