Glioma is recognized as a highly angiogenic malignant brain tumor. Vasculogenic mimicry (VM) greatly restricts the therapeutic effect of anti-angiogenic tumor therapy for glioma patients. However， the molecular mechanisms of VM formation in glioma remain unclear. Here， we demonstrated that LINC00339 was upregulated in glioma tissue as well as in glioma cell lines. The expression of LINC00339 in glioma tissues was positively correlated with glioma VM formation. Knockdown of LINC00339 inhibited glioma cell proliferation， migration， invasion， and tube formation， meanwhile downregulating the expression of VM-related molecular MMP-2 and MMP-14. Furthermore， knockdown of LINC00339 significantly increased the expression of miR-539-5p. Both bioinformatics and luciferase reporter assay revealed that LINC00339 regulated the above effects via binding to miR-539-5p. Besides， overexpression of miR-539-5p resulted in decreased expression of TWIST1， a transcription factor known to play an oncogenic role in glioma and identified as a direct target of miR-539-5p. TWIST1 upregulated the promoter activities of MMP-2 and MMP-14. The in vivo study showed that nude mice carrying tumors with knockdown of LINC00339 and overexpression of miR-539-5p exhibited the smallest tumor volume through inhibiting VM formation. In conclusion， LINC00339 may be used as a novel therapeutic target for VM formation in glioma.