Abstract Chitinase-3-like protein 1 (CHI3L1) plays multiple roles in inflammation, tissue remodelling, and wound healing. It is a new, potentially druggable target for the treatment of cancer due to its association with both extracellular matrix (ECM) modifications and potential involvement in neoangiogenesis. Expression of CHI3L1 is upregulated in glioblastoma (GBM), the most common and aggressive primary brain tumour. The role of CHI3L1 in GBM progression is not fully understood and calls for further research. We generated a knockout (KO) of CHI3L1 in human glioblastoma U87-MG cells. To evaluate the effects of CHI3L1 KO on tumour growth, wild-type (WT) or CHI3L1 KO cells were intracranially implanted into athymic mice and tumour volume was assessed with magnetic resonance imaging. We have measured the serum level of CHI3L1 in both groups and correlated it with the tumour burden. Immunofluorescent staining against endothelium marker – von Willebrand factor (vWF), and perivascular water channel – aquaporin 4 (AQP4) were employed to evaluate the tumour vasculature in both groups. We found that CHI3L1 KO in glioblastoma cells significantly reduced volume of established tumors. Blood serum level of CHI3L1 positively correlated with tumour burden of experimental animals, which corroborates its role as a biomarker. Immunostaining for vWF revealed that in CHI3L1 KO gliomas there were more non-capillary blood vessels than in controls. We observed a higher level of AQP4 in CHI3L1 KO tumours, which suggests a difference in the structure of perivascular space, and thus vasculature functionality, in CHI3L1 depleted gliomas. In conclusion, we demonstrated the involvement of glioma-derived CHI3L1 in the growth and neovasculature of experimental human gliomas. Our findings suggest that the depletion of CHI3L1 in glioma cells results in a higher number and possibly different structure of non-capillary blood vessels in the tumour. Studies supported by the grant N 2020/39/B/NZ4/02683 from the National Science Center.