Abstract Diacylglycerol kinase (DGK) is a lipid kinase that catalyzes the phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA), which uses ATP as a phosphate donor. Diacylglycerol kinases ζ(DGKζ) is characterized as specific type IV due to its myristoylated alanine-rich C-kinase substrate (MARCKS), ankyrin, and PDZ binding domain. Similar to other DGKs, DGKζ is also reported to be abnormally expressed in human colorectal cancer cells, and it is indispensable for the proliferation of cancer cells. However, its implications in human glioblastoma (GBM) is largely unknown. Both the mRNA and protein levels of DGKζ were significantly higher in GBM tissues than in precancerous lesions. Knockdown of DGKζ inhibited GBM cell proliferation, cell cycle and promoted apoptosis of GBM cells. Moreover, down-regulation of DGKζ markedly reduced in vitro colony formation and in vivo tumorigenic capability. Furthermore, we confirmed that DGKζ was the downstream target of miR-34a. The expression level of DGKζ was negatively correlated with miR-34a in GBM tissues. Overexpression of DGKζ reversed the tumor suppressive roles of miR-34a in GBM cells. Taken together, DGKζ can act as a potential prognostic biomarker for GBM patients and promote the growth of GBM cells was regulated by miR-34a, and it may represent a promising therapeutic target for patients with GBM.