Glioblastoma (GBM) is the most common primary central nervous system tumor and has a poor prognosis, with a median survival time of only 14 months from diagnosis. Abnormally expressed long noncoding RNAs (lncRNAs) are important epigenetic regulators of chromatin modification and gene expression regulation in tumors, including GBM. We previously showed that the lncRNA HOTAIR is related to the cell cycle progression and can be used as an independent predictor in GBM. Lysine-specific demethylase 1 (LSD1), binding to 3' domain of HOTAIR, specifically removes mono- and di-methyl marks from H3 lysine 4 (H3K4) and plays key roles during carcinogenesis. In this study, we combined a HOTAIR-EZH2 disrupting agent and an LSD1 inhibitor, AC1Q3QWB (AQB) and GSK-LSD1, respectively, to block the two functional domains of HOTAIR and potentially provide therapeutic benefit in the treatment of GBM. Using an Agilent Human ceRNA Microarray, we identified tumor suppressor genes upregulated by AQB and GSK-LSD1, followed by Chromatin immunoprecipitation (ChIP) assays to explore the epigenetic mechanisms of genes activation. Microarray analysis showed that AQB and GSK-LSD1 regulate cell cycle processes and induces apoptosis in GBM cell lines. Furthermore, we found that the combination of AQB and GSK-LSD1 showed a powerful effect of inhibiting cell cycle processes by targeting CDKN1A, whereas apoptosis promoting effects of combination therapy were mediated by BBC3 in vitro. ChIP assays revealed that GSK-LSD1 and AQB regulate P21 and PUMA, respectively via upregulating H3K4me2 and down-regulating H3K27me3. Combination therapy with AQB and GSK-LSD1 on tumor malignancy in vitro and GBM patient-derived xenograft (PDX) models shows enhanced anti-tumor efficacy and appears to be a promising new strategy for GBM treatment through its effects on epigenetic regulation.