Long non-coding RNAs (lncRNAs) have been recently shown to be dysregulated and closely related to several cancers. Here, we aimed to elucidate the function and the possible molecular mechanisms of lncRNA Metastasis-associated lung Adenocarcinoma transcript-1 (MALAT1) in human glioma. Quantitative real-time PCR (qRT-PCR) was used to detect the expressions of MALAT1, miR-101 and Rap1B mRNA in U251 and U87 cells. The protein level of Rap1B was examined by western blot assays. Moreover, the proliferation and apoptosis of U251 and U87 cells were determined by CCK-8 assay and flow cytometry analysis, respectively. Additionally, the targets of miR-101 were identified by target prediction and luciferase reporter assays. The results demonstrated that MALAT1 and Rap1B were upregulated, while miR-101 expression was downregulated in glioma cell lines U251 and U87. MALAT1 and Rap1B knockdown could inhibit proliferation and induce apoptosis of glioma cells. Moreover, MALAT1 promoted the Rap1B expression by sponging miR-101 in U251 and U87 cells. Furthermore, miR-101 downregulation or Rap1B overexpression reversed the proliferation inhibitory and apoptosis induction of glioma cell lines caused by MALAT1 knockdown. Taken together, MALAT1 promotes proliferation and suppresses apoptosis of glioma cells through derepressing Rap1B by sponging miR-101. The present study elucidates a novel MALAT1-miR-101-Rap1B regulatory axis in glioma, contributing to a better understanding of the glioma pathogenesis and providing a promising therapeutic target for glioma patients.