In this study, we examined the unique relationship of maspin, a serine protease inhibitor (serpin), that plays a critical role in mammary gland development and is silenced during breast cancer progression, and nitric oxide (NO), a multifaceted water and lipid soluble free radical. The hypothesis tested was that there is a correlation between endothelial nitric oxide synthase and maspin in MCF-7 cells and that NO is capable of regulating maspin expression. An experimental system was developed in which cellular levels of NO in normal human mammary epithelial cells and the breast cancer cell line MCF-7 could be altered using NO modulators. The effect(s) of NO modulators on maspin was measured using reverse transcriptase-polymerase chain reaction and Western blot analysis of subcellular fractions of both cell types. The data revealed that NO induced maspin expression in MCF-7 cells, and the induced maspin resulted in diminished cell motility and invasion, concomitant with an increase in the apoptotic index. This novel finding provides new information regarding the molecular role of maspin in regulating mammary epithelial growth, remodeling, tumor progression, and the metastatic process. More significantly, these findings could have a potential impact on future therapeutic intervention strategies for breast cancer. Targeted delivery of NO within the tumor microenvironment could provide a feasible noninvasive approach for effective treatment.