The constitutive activation of signal transducer and activator of transcription 3 (Stat3) is frequently detected in breast cancer cell lines but not in normal breast epithelial cells. Stat3 has been classified as an oncogene, because constitutively active Stat3 can mediate oncogenic transformation in cultured cells and tumor formation in nude mice. Since Stat3 appears to play an important role in breast cancer, it is of interest to investigate Stat3-regulated genes and elucidate Stat3-mediated oncogenesis. In this study, we investigated the Stat3-regulated genes in human breast epithelial cells. Upon overexpression of Stat3-C, a constitutively active Stat3 form, in nonmalignant telomerase immortalized breast (TERT) cells, the total mRNA was extracted and subjected to Affymetrix microarray analysis. Our results showed that mitogen-activated protein kinase kinase 5 (MEK5) was markedly induced (more than 22-fold increase, P<0.001) by Stat3-C expression. RT-PCR result also demonstrated that MEK5 mRNA was significantly induced by Stat3-C in TERT cells. The upregulation of MEK5 by Stat3-C was further confirmed by Western blot in MCF10A breast epithelial cells. Furthermore, in MDA-MB-435s breast carcinoma cells, which express high levels of activated Stat3 and MEK5, MEK5 protein was significantly reduced by using Stat3 short interfering RNA. The reduction of MEK5 was consistent with Stat3 knockdown in this breast carcinoma cell line. We also investigated MEK5 expression in different breast carcinoma cell lines and breast cancer tissues using tissue array analysis. Compared with nonmalignant breast epithelial cells or normal tissues without constitutively active Stat3 signaling, MEK5 protein levels are remarkably higher in breast carcinoma cell lines and cancer tissues with constitutively activated Stat3. Taken together, our findings suggest that constitutively active Stat3 upregulates MEK5 in the breast epithelial cells. MEK5 may be one of the Stat3-regulated genes and plays its essential roles in oncogenesis mediated by aberrantly activated Stat3 signaling in breast carcinomatosis and malignancies.