Eph receptor tyrosine kinases mediate neurodevelopmental processes such as boundary formation, vasculogenesis, and cell migration. Recently, we found that overexpression of EphB2 in glioma cells results in reduced cell adhesion and increased cell invasion. Since R-Ras has been shown to play a critical role in EphB2 regulation of integrin activity, we explored whether the biological role of EphB2 in glioma invasion is mediated by downstream R-Ras activation. On EphB2 activation, R-Ras associated with the receptor and became highly phosphorylated. Depletion of endogenous R-Ras expression by siRNA abrogated EphB2 effects on glioma cell adhesion, proliferation, and invasion in ex vivo rat brain slices. Anti-proliferative responses to EphB2 activation were consistent with suppressed mitogen-activated protein kinase activity. Moreover, R-Ras was highly phosphorylated in the invading glioma cells. In human brain tumor specimens, R-Ras expression and phosphorylation correlated with increasing grade of gliomas. Laser capture microdissection of invading glioblastoma cells revealed elevated R-Ras mRNA (1.5- to 26-fold) in 100% (eight of eight) of biopsy specimens, and immunohistochemistry revealed high R-Ras localization primarily in glioblastoma cells. The phosphorylation ratio of R-Ras positively correlated with the phosphorylation ratio of EphB2 in glioblastoma tissues. These results demonstrate that R-Ras plays an important role in glioma pathology, further suggesting the EphB2/R-Ras signaling pathway as a potential therapeutic target.