EFA6A, or Pleckstrin and Sec7 domain protein, is a member of guanine nucleotide exchange factors for ADP ribosylation factor 6 (ARF6). Whereas EFA6A is specifically expressed in the brain, little is known about its function in glial cells or glioma. Here we show that elevated EFA6A expression is detectable in both low-grade and high-grade human glioma tissues samples. To investigate the role of EFA6A in glioma carcinogenesis, we generated a human glioblastoma cell line which conditionally overexpresses EFA6A (U373-EFA6A). We showed that overexpression of EFA6A had no effect on cell proliferation, apoptosis, or cell cycle control. However, as shown by wound healing and in vitro cell invasion assays, it significantly enhanced the cell motility and invasiveness whereas silencing EFA6A by its dominant negative mutant EFA6A(E242K) produced opposite effects. We further showed that ARF6/extracellular signal-regulated kinase (ERK) signaling is required for the EFA6A-mediated cell invasion because both EFA6A(E242K) and ARF6 dominant negative mutant ARF6(T27N) markedly reduced the phosphorylated ERK level and EFA6A-mediated invasive capacity. Consistently, mitogen-activated protein kinase/ERK kinase inhibitor U0126 could abolish the EFA6A-induced cell invasion. These results suggest for the first time a potential role of EFA6A/ARF6/ERK signal cascade in glioma cell migration and invasion.