Cyclooxygenase-2 (COX-2) is frequently overexpressed in human cancers and contributes to the malignant phenotype. Our data indicate unphosphorylated signal transducers and activators of transcription 6 (STAT6) may transcriptionally upregulate COX-2 expression and protect against apoptosis in NSCLC cells. In A427 and H2122, NSCLC cell lines that constitutively express COX-2, only unphosphorylated STAT6 was detectable by western blot, thus, all of the following STAT6-dependent effects are attributed to the unphosphorylated protein. In both cell lines, small-interfering RNA-mediated knockdown of STAT6 or stable expression of dominant-negative STAT6 decreased COX-2 expression. In contrast, transfection with a phosphorylation-deficient mutant STAT6 increased COX-2 levels. Immunofluorescent staining revealed the presence of STAT6 in H2122 nuclei, suggesting a direct role in gene regulation for the unphosphorylated protein. Consistent with this hypothesis, unphosphorylated STAT6 increased luciferase expression from a COX-2 promoter reporter construct. STAT6 co-immunoprecipitated with the transcriptional co-activator, p300, and chromatin immunoprecipitation assays demonstrated that these proteins bind a consensus STAT6 binding site located within the COX-2 promoter. STAT6 DNA-binding specificity was confirmed by electrophoretic mobility shift assay. As COX-2 over-expression has been clearly linked to apoptosis resistance and other hallmarks of malignancy, these findings suggest a novel role of unphosphorylated STAT6 in the pathogenesis of non-small cell lung cancer.