Background: Glioblastoma (GBM) is one of the most aggressive and malignant tumor types. Despite treatment advances, GBM pathogenesis still remains largely unknown. MATN1-AS1, an intron-retained long non-coding RNA (lncRNA), has been implicated in GBM development. However, the underlying mechanism has not been identified. This study aimed to examine MATN1-AS1 expression and uncover its role in GBM. Methods: LncRNAs with low expression levels were selected by analyzing brain glioma-related genes. The relative mRNA level of MATN1-AS1 was quantified using RT-qPCR in 75 GBM tumors and 10 normal brain tissues. Overall survival was assessed using the Kaplan-Meier method. RT-qPCR and immunoblotting analysis were carried out to assess the levels of MATN1-AS1, RELA, ERK1/2, Bcl-2, Bax, survivin, and MMP-9 in GBM cells. Biological functions of MATN1-AS1 in GBM tumors were measured both in vivo and in vitro. The mechanism of RELA regulation by MATN1-AS1 was detected using RNA pull-down, RNA-binding protein immunoprecipitation, chromatin immunoprecipitation, and the dual luciferase reporter gene assay. Results: MATN1-AS1 was the most downregulated lncRNA in GBM and was correlated with a shorter survival time and poorer prognosis of GBM patients. Conversely, RELA was increased in GBM tumor tissues and negatively correlated with MATN1-AS1 expression. MATN1-AS1 over-expression or siRNA-RELA knockdown resulted in downregulation of mRNA and protein levels of RELA, ERK1/2, Bcl-2, survivin, and MMP-9; reduced cell proliferation and invasion; increased Bax mRNA and protein levels; and enhanced cellular apoptosis. MATN1-AS1 bound to E2F6, which negatively targeted RELA. Furthermore, MATN1-AS1 over-expression in GBM cells resulted in significant inhibition of tumor growth in vivo. Conclusions: Upregulation of the lncRNA MATN1-AS1 inhibited GBM cell proliferation and invasion through inhibition of RELA via E2F6 and suppression of the MAPK signaling pathway. MATN1-AS1 might be an underlying therapeutic target for GBM.