Glioblastoma multiforme (GBM) is the most malignant and frequent brain tumor, with an aggressive growth pattern and poor prognosis despite best treatment modalities. Although chemotherapy with temozolomide (TMZ) may restrain tumor growth for some months, TMZ resistance is also common and accounts for many treatment failures. Research into microRNA's role in GBM has shown that microRNAs play a key regulatory role in the GBM, making it a potential therapeutic target. In this study, we demonstrated that the lower expression of miR-181a/b/c/d subunits contributes to astrocytoma tumorigenesis, and their overexpression could inhibit the invasive proliferation of glioblastoma cells by targeting Rap1B-mediated cytoskeleton remodeling and related molecular (Cdc42, RhoA and N-cadherin) changes, suggesting that miR-181 was a critical regulator and might be an important target for glioblastoma treatment. TMZ as a standard chemotherapeutic agent for GBM inhibited the Rap1B expression and actin cytoskeleton remodeling to exert its cell killing by upregulating miR-181a/b/c/d subunits; conversely, each miR-181a/b/c/d subunit enhanced the chemosensitivity of TMZ in glioblastoma cells.