Maspin, a unique serine proteinase inhibitor (serpin), plays a key role in mammary gland development and is silenced during breast cancer progression. Maspin has been shown to inhibit tumor cell motility and invasion in cell culture, as well as growth and metastasis in animal models. In this study, we investigated the effect of maspin on the regulation of hypoxia-induced expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), with respect to invasive potential in metastatic breast cells MDA-MB-231. We hypothesized that maspin can neutralize or mitigate hypoxia-induced expression of uPA/uPAR in metastatic breast cancer cells, resulting in suppression of their invasive potential. To test our hypothesis, we employed the highly invasive MDA-MB-231 breast cancer cells that are devoid of maspin, and transfected them with the maspin gene, and then determined the effect of hypoxia on uPA/uPAR expression. Normal mammary epithelial cells 1436N1 were used as a control. Our findings demonstrate that maspin downregulated the basal and hypoxia-induced uPA/uPAR expression and reduced the stimulatory effect of hypoxia on the in vitro invasive ability of MDA-MB-231-cells. In addition, maspin also inhibited the enzymatic activity of secreted and cell associated uPA in MDA-MB-231 cells. These results indicate that maspin inhibits hypoxia-induced invasion of metastatic breast cancer cells by blocking the uPA system, thus illuminating an important molecular pathway for therapeutic consideration.