Glioblastoma is diagnosed by biopsy or, if clinically feasible, tumor resection. However, emerging evidence suggests that this surgical intervention may increase the risk of tumor cell spread. It has been hypothesized that the damage to the tumor leads to infiltration of immune cells that consequently form an environment that favors tumor cell motility. In mouse glioma models, it was previously found that biopsy induced migration of tumor cells in vivo and that recruitment of monocytes from the blood was involved in this effect. However, the role of neutrophils in this process is still unclear. Here, we study the contribution of neutrophils on the pro-migratory effect of surgical interventions in glioma. Using repetitive intravital microscopy, in vivo migration of glioma tumor cells before and after biopsy was compared in mice systemically depleted of neutrophils. Interestingly, macrophages/microglia were almost completely absent from neutrophil-depleted tumors, indicating that neutrophils may be indirectly involved in biopsy-induced migration of glioma tumor cells through the recruitment of macrophages to the tumor. To further investigate whether neutrophils have the potential to also directly promote glioblastoma tumor cell migration, we performed in vitro migration assays using human neutrophils. Indeed, wound-healing of human primary glioblastoma tumor cell lines was promoted by human neutrophils. The pro-migratory effects of human neutrophils on glioblastoma tumor cells could also be recapitulated in transwell migration assays, indicating that soluble factor(s) are involved. We therefore provide evidence for both an indirect and direct involvement of neutrophils in tumor spread following biopsy of glioblastoma tumors.