Abstract Emerging evidence indicate novel modes of EGFR signaling, notably, one involves EGFR nuclear translocalization and subsequent gene activation. To date, however, the significance of the nuclear EGFR pathway in glioblastoma (GBM), the most frequent and deadliest brain malignancy in adults, is unknown. Here, we report that EGFR and its constitutively activated variant EGFRvIII undergo nuclear translocalization in GBM cells, in which the former event requires EGF stimulation and the latter is constitutive. To gain insight into the impact of nuclear EGFR on gene expression in GBM, we created isogenic GBM cell lines, namely, U87MG-vector, U87MG-EGFR and U87MG-EGFRdNLS that, respectively, express the control vector, EGFR and nuclear-entry defective EGFR with a deletion of the nuclear localization signal (NLS). Microarray analysis shows that 19 genes, including, cyclooxygenase-2 (COX-2), to be activated in U87MG-EGFR cells but not in U87MG-EGFRdNLS and U87MG-vector cells. Subsequent validation studies indicate that COX-2 gene is expressed at higher levels in cells with EGFR and EGFRvIII than those with EGFRdNLS and EGFRvIIIdNLS. Nuclear EGFR and its transcriptional co-factor STAT3 associate with the COX-2 promoter. Increased expression of EGFR/EGFRvIII and activated STAT3 leads to synergistic activation of the COX-2 promoter. Promoter mutational analysis identified a proximal STAT3-binding site that is required for EGFR/EGFRvIII-STAT3 mediated COX-2 gene activation. In GBM tumors, an association exists between levels of COX-2, EGFR/EGFRvIII and activated STAT3. Together, these findings indicate the existence of the nuclear EGFR/EGFRvIII signaling pathway in GBM and its functional interaction with STAT3 to activate COX-2 gene expression, thus linking EGFR-STAT3 and EGFRvIII-STAT3 signaling axes to pro-inflammatory COX-2 mediated pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3117.