Nogo or reticulon-4 (RTN4), also known as neurite outgrowth inhibitor, is a member of the reticulon family of genes. Nogo-A, one of the three isoforms, is enriched in the central nervous system (CNS). The extracellular domain of Nogo-A, Nogo-66, has neurite growth inhibitory activity that is specific for neurons and is mediated by the Nogo receptor. However, most of its functions are not known yet. We investigated whether Nogo-A modulates the migration and invasion of a glioblastoma cell line, as well as the factors that have an effect on Nogo-A. The expression of Nogo-A was evaluated using western blotting and immunohistochemistry in human brain tumor specimens. U87MG cells were transfected with a sense-Nogo-A cDNA construct (U87-Nogo-A cells expressing Nogo-A) and an empty vector (U87MG-E cells not expressing Nogo-A). The migration and invasion abilities of these cells were investigated using simple scratch and Matrigel invasion assays. Morphologic and cytoskeletal changes were documented by confocal microscopy. The proliferation rate was estimated using doubling time assay. The effects of Nogo-A on Rho activity and phosphorylated cofilin were determined by a Rho activity assay and western blotting. Among primary brain tumors, Nogo-A expression was found in a higher percentage of oligodendrogliomas (90.0%) compared with the percentage in the glioblastomas (68.4%). In addition, the percentage in mixed gliomas was 42.9%, while it was not expressed in pituitary adenomas or schwannomas. The migration and invasion abilities of the U87-Nogo-A cells were decreased compared with the control. In the U87-Nogo-A cell line, Rho activity and phosphorylated cofilin expression were also decreased and morphology became more flat in comparison with the U87MG-E cell line. Nogo-A may inhibit the migration and invasion of human malignant glioma cells via the downregulation of RhoA-cofilin signaling.