Glioblastoma (GBM) is the most common primary malignant brain cancer in adults. A hallmark of GBM is aggressive invasion of tumor cells into the surrounding normal brain. The current standard of care therapy, as well as targeted therapies, have largely failed to specifically address this issue. Therefore, identifying key regulators of GBM cell migration and invasion is of particular interest. The leukemia-associated RhoGEF (LARG) has previously been implicated in cell invasion in other tumor types; however, the role of LARG in GBM pathobiology remains undefined. Here, we report that the expression level of LARG, RhoC, and RhoA increases with glial tumor grade and is highest in GBM. LARG and RhoC protein expression is more prominent in the invading cells whereas RhoA expression is largely restricted to cells in the tumor core. Knockdown of LARG by siRNA inhibits GBM cell migration in vitro and invasion ex vivo in organotypic brain slices. Moreover, siRNA mediated silencing of RhoC suppresses GBM cell migration in vitro and invasion ex vivo, whereas depletion of RhoA enhances GBM cell migration and invasion, supporting a role for LARG and RhoC in GBM cell migration and invasion. Depletion of LARG increases the sensitivity of GBM cells to temozolomide treatment. Collectively, these results suggest that LARG and RhoC may represent unappreciated targets to inhibit glioma invasion.Copyright © 2020. Published by Elsevier Inc.