Glioblastoma is extremely lethal brain cancer. In our research, we investigated the effect of anti-vimentin nanobody Nb79 on glioblastoma cell invasion. The expression of vimentin in glioma tissues and cells was determined by RT-qPCR. Invasion assay was performed on differentiated glioblastoma cell line U-87 MG and stem cell line NCH421k in vitro and in vivo in zebrafish embryos. The effect of Nb79 on expression of EMT biomarkers was determined by Western blot and immunocytochemistry. Our study shows that vimentin is upregulated in glioblastoma tissue compared to lower grade glioma and non-tumour brain tissue. Nb79 reduced glioblastoma cell invasion by up to 64% in vitro and up to 21% in vivo. The tight junction protein ZO-1 had higher expression on the cell membrane, when treated with Nb79 compared to control. In conclusion, our results suggest that anti-vimentin nanobody is a promising tool to target glioblastoma cell invasion.