Dipeptidyl-peptidase III (DPP III) is a cytosolic metallo-aminopeptidase implicated in various physiological and pathological processes. A previous study from our laboratory indicated an elevated expression of DPP III in glioblastoma (U87MG) cells. In the present study we investigated the role of interleukin-6 (IL-6), a pleiotropic cytokine produced by glial tumors, in the regulation of DPP III expression. Immunohistochemistry, western blotting and quantitative RT-PCR were used for quantitation of DPP III and IL-6 in human glioblastoma cells and tumors. Cell transfections and DPP III promoter reporter assays were performed to study the transcriptional regulation of DPP III by IL-6. Promoter deletion analysis, site directed mutagenesis, chromatin immunoprecipitation assays and small interfering RNA (siRNA) technology was employed to elucidate the molecular mechanism of IL-6 mediated regulation of DPP III expression in glioblastoma cells. Our results for the first time demonstrate a negative correlation (r = 0.632, P = 0.01) between DPP III and IL-6 in both human tumors and cultured glioblastoma cells. Treatment of U87MG cells with IL-6 significantly decreased DPP III expression with a concomitant increase in the levels of transcription factor CCAAT/enhancer binding protein beta (C/EBP-β). Deletion/mutagenesis of C/EBP-β binding motif of DPP III promoter significantly increased its activity and abolished its responsiveness to IL-6. This effect could also be mimicked by C/EBP-β siRNA. In conclusion our study for the first time demonstrates C/EBP-β mediated transcriptional downregulation of DPP III by IL-6. Our results demonstrating a negative correlation between IL-6 and DPP III taken together with the previously reported prognostic significance of this cytokine in glioblastoma suggests that DPP III may prove useful as a prognostic marker.