Blocking migration is an attractive strategy to inhibit glioma tumorigenesis. Previous studies have indicated that neomycin inhibits glioma cells proliferation. The purpose of this study was to expand on the preliminary research by investigating the antimigratory effect of neomycin. We used glioma C6 cells to investigate the role of neomycin in cell migration in vitro. Cell wounding assay showed that neomycin inhibits in a dose-dependent manner glioma cells migration into the wound. Taken together, these results suggest that neomycin is an attractive candidate for the development of novel antimigratory molecules useful for the treatment of gliomas.