Rho GDP dissociation inhibitor beta (Rho-GDI beta), an inhibitor of Rho GTPases, is primarily expressed by hematopoietic cells but is also found in epithelial cancer cells. Recently, we have identified Rho-GDI beta as a target of the transcription factor Ets1. Here, we show that, in breast cancer cells, Ets1 regulates Rho-GDI beta expression and binds to the upstream region of the Rho-GDI beta gene. Furthermore, in primary breast cancer, Rho-GDI beta is coexpressed with Ets1. Studying the function of Rho-GDI beta in breast cancer, we found that a Rho-GD beta-specific small interfering RNA increased cellular migration but also decreased the expression of cyclooxygenase-2 (Cox-2) oncogene as shown by microarray, quantitative reverse transcription-PCR, and Western blot analyses. Further studies revealed that Rho-GDI beta regulates Cox-2 gene at least partly on the transcriptional level, most likely by activating nuclear factor of activated T cells 1 (NFAT-1). Vav-1, an interaction partner of Rho-GDI beta, was also found to interfere with Cox-2 expression and NFAT-1 cellular distribution, suggesting a cooperative action of Rho-GDI beta and Vav-1 on Cox-2 expression. To explore the importance of Rho-GDI beta for the survival of breast cancer patients, two cohorts, including 263 and 117 patients, were analyzed for clinical outcome in relation to Rho-GDI beta RNA and protein levels, respectively. Expression of Rho-GDI beta was not associated with either disease-free or overall survival in the two patient population. Our data suggest that the expression of Rho-GDI beta in breast cancer is neither beneficial nor disadvantageous to the patient. This may be the net effect of two opposing activities of Rho-GDI beta, one that suppresses tumor progression by inhibiting migration and the other that stimulates it by enhancing Cox-2 expression.