Glioblastoma (GBM) is the most common and fatal primary tumor of the central nervous system (CNS) and current treatments have limited success. Chemokine signaling regulates both malignant cells and stromal cells of the tumor microenvironment (TME), constituting a potential therapeutic target against brain cancers. Here, we investigated the C–C chemokine receptor type 7 (CCR7) and the chemokine (C–C-motif) ligand 21 (CCL21) for their expression and function in human GBM and then assessed their therapeutic potential in preclinical mouse GBM models. In GBM patients, CCR7 expression positively associated with a poor survival. CCL21–CCR7 signaling was shown to regulate tumor cell migration and proliferation while also controlling tumor associated microglia/macrophage recruitment and VEGF-A production, thereby controlling vascular dysmorphia. Inhibition of CCL21–CCR7 signaling led to an increased sensitivity to temozolomide-induced tumor cell death. Collectively, our data indicate that drug targeting of CCL21–CCR7 signaling in tumor and TME cells is a therapeutic option against GBM.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-023-04788-7.