Glioblastoma multiforme (GBM) is the most common in situ neoplasms in central nervous system (CNS). However, the pathogenesis of GBM is poorly understood. Long noncoding RNAs (lncRNAs) have been implicated in GBM progression. In this study, we attempted to identify the biological role of the EGFR-AS1 in glioma cells and tissues, as well as reveal the molecular mechanism associated. The results indicated that lnc-EGFR-AS1 expression was increased in glioma cells and tissues. EGFR-AS1 knockdown suppressed proliferation, migration and invasion of glioma cells, but induced apoptosis. Additionally, lnc-EGFR-AS1 functioned as a sponge for miR-133b. Promoting lnc-EGFR-AS1 expression significantly reduced miR-133b expression. Furthermore, miR-133b could target the 3′-untranslated region (3′-UTR) of RACK1 and reduced its expression levels. What’s more, lnc-EGFR-AS1 knockdown reduced RACK1 expression partly through enhancing miR-133b expression. In vivo experiments confirmed the anti-tumorigenesis capability of EGFR-AS1 knockdown. These findings elucidated that EGFR-AS1 accelerated cell proliferation, migration, invasion and prevented apoptosis in glioma cells by regulating miR-133b/RACK1, providing new insights for the diagnosis and molecular therapy of GBM.