Here, we found that ZEB1-AS1 was highly expressed in glioma tissues, being closely related to clinical stage of glioma. Functionally, silencing of ZEB1-AS1 could significantly inhibit cell proliferation, migration, and invasion, as well as promote apoptosis. Knockdown of ZEB1-AS1 significantly induced the G0/G1 phase arrest and correspondingly decreased the percentage of S phase cells. Further analysis indicated that ZEB1-AS1 could regulate the cell cycle by inhibiting the expression of G1/S transition key regulators, such as Cyclin D1 and CDK2. Furthermore, ZEB1-AS1 functioned as an important regulator of migration and invasion via activating EMT through up-regulating the expression of ZEB1, MMP2, MMP9, N-cadherin, and Integrin-B1 as well as decreasing E-cadherin levels in the metastatic progression of glioma.