Glioblastoma (GBM) is one of the most aggressive primary brain tumors with frequent recurrences following the standard methods of treatment—temozolomide (TMZ), ionizing radiation and surgical resection. The objective of our study was to investigate GBM resistance mediated via MMP14 (matrix metalloproteinase 14). We used multiple PDX GBM models and established glioma cell lines to characterize expression and subcellular localization of MMP14 after TMZ treatment. We performed a Kiloplex ELISA‐based array to evaluate changes in cellular proteins induced by MMP14 expression and translocation. Lastly, we conducted functional and mechanistic studies to elucidate the role of DLL4 (delta‐like canonical notch ligand 4) in regulation of glioma stemness, particularly in the context of its relationship to MMP14. We detected that TMZ treatment promotes nuclear translocation of MMP14 followed by extracellular release of DLL4. DLL4 in turn stimulates cleavage of Notch3, its nuclear translocation and induction of sphering capacity and stemness.