Nogo-66 is a 66-amino-acid-residue extracellular domain of Nogo-A, which plays a key role in inhibition neurite outgrowth of central nervous system through binding to the Nogo-66 receptor (NgR) expressed on the neuron. Recent studies have confirmed that NgR is also expressed on the surface of macrophages/microglia in multiple sclerosis, but its biological effects remain unknown. In the present study, our results demonstrated that Nogo-66 triggered microglia anti-adhesion and inhibited their migration in vitro, which was mediated by NgR. We also assessed the roles of small GTP (glycosyl phosphatidylinositol)-binding proteins of the Rho family as the downstream signal transducers on the microglia adhesion and mobility induced by Nogo-66. The results showed that Nogo-66 activated RhoA and reduced the activity of Cdc42 in the meanwhile, which further triggered the anti-adhesion and migration inhibition effects to microglia. Nogo-66 inhibited microglia polarization and membrane protrusion formation, thus might eventually contribute to the decreasing capability of cell mobility. Taken together, the Nogo-66/NgR pathway may modulate neuroinflammation via mediating microglia adhesion and migration in addition to its role in neurons. Better understanding the relationship between Nogo-66/NgR and neuroinflammation may help targeting NgR for treating central nervous system diseases related with inflammation.