RET finger protein (RFP) forms a complex with histone deacetylase 1, resulting in aberrant deacetylation of H3K27ac and dysregulation of cis-regulatory elements. We evaluated the modulatory effects of RFP knockdown on cis-regulatory elements, gene expression, and chemosensitivity to temozolomide both in glioblastoma cells and in an intracranial glioblastoma model. The combination of RFP knockdown and temozolomide treatment markedly suppressed the glioblastoma cell growth due to oxidative stress and aberrant cell cycle and increased survival time in mice with glioblastoma. ChIP-seq and RNA-seq revealed that RFP knockdown increased or decreased activity of numerous cis-regulatory elements that lie adjacent to genes that control functions such as apoptosis, mitosis, DNA replication, and cell cycle: FOXO1, TBP2, and PARPBP. This study suggests that RFP contributes to chemoresistance via aberrant deacetylation of histone H3 at K27, whereas dysregulation of RFP-associated cis-regulatory elements in glioma and RFP knockdown combined with temozolomide is an effective treatment strategy for lethal glioma. Display omitted •RET finger protein (RFP) binds to histone deacetylase 1 in glioblastoma cells•RFP regulates H3K27acetylation and cis-regulatory elements•Knockdown of RFP modulates elements of oxidative stress and base excision repair•Knockdown of RFP overcomes chemoresistance in glioblastoma in vitro and in vivo Ranjit et al. demonstrate that the RET finger protein (RFP), which forms a complex with histone deacetylase 1 in glioma cells, also alters H3K27 acetylation patterns and cis-regulatory elements. Furthermore, RFP knockdown regulates the factors of oxidative stress and base excision repair, thereby enhancing temozolomide chemosensitivity in glioblastoma.