BACKGROUND: Resistance of glioblastoma to standard chemoradiotherapy may be mediated, at least partially, by a population of inherently resistant glioma stem cells (GSCs) which are able to survive treatment and reconstitute the tumour. HOX genes are dysregulated in various cancers and a subset of GSCs show high levels of HOX expression. In addition, a HOX gene signature has been associated with treatment resistance in glioblastoma. Our goal was to determine the potential of targeting HOX function as a novel therapy for glioblastoma. METHODS: HOX expression profiles for patient-derived GSCs and established glioma cell lines were determined by quantitative RT-PCR. In vitro, cells were treated with the HOX inhibitor peptide HXR9 (or control peptide CXR9), with or without radiation two hours later. Subsequently, cells were incubated through two doubling times and the MTS assay was used to determine cell viability. In vivo, sub-cutaneous xenografts of U87-MG cells were established in Balb/c Nude mice. Treatments (peptide and/or radiation) or controls were administered in three doses over consecutive days. RESULTS: Analysis of HOX expression in GSC lines demonstrated up-regulation of genes across all four clusters, although expression patterns varied between cell lines. In contrast, the established glioma cell lines T98G and U87-MG showed lower overall HOX expression. The HOX inhibitor peptide HXR9 had potent cytotoxic activity in a number of GSC lines, with IC50 values between 3 and 5 µM. In contrast, T98G and U87-MG were more resistant (IC50s of 70 and 35 µM, respectively), as was a normal neural progenitor cell line NP1 (IC50 65 µM). In vitro, combinations of sub-lethal doses of HXR9 with radiation demonstrated an additive cytoxic effect on GSCs. In vivo, treatment with HXR9 peptide resulted in tumour growth delay when combined with radiotherapy in a mouse xenograft model. CONCLUSIONS: Knockdown of HOX protein function reduced viability of glioma cells in vitro, with greater potency in GSCs than established cell lines. Combination of HOX-inhibition with radiation or demonstrated an additive cytoxicity in vitro and improved tumour growth delay in a preclinical mouse model. Therefore, targeting HOX proteins shows potential as an adjuvant therapy with current standard of care chemoradiotherapy for glioblastoma.