Mitochondrial dysfunction is a hallmark of cancer biology. Tumor mitochondrial metabolism is characterized by an abnormal ability to function in scarce oxygen conditions through glycolysis (the Warburg effect), and accumulation of mitochondrial DNA defects are present in both hereditary neoplasia and sporadic cancers. Mitochondrial Lon is a major regulator of mitochondrial metabolism and the mitochondrial response to free radical damage, and plays an essential role in the maintenance and repair of mitochondrial DNA. Despite these critical cellular functions of Lon, very little has been reported regarding its role in glioma. Lon expression in gliomas and its relevance with patient survival was examined using published databases and human tissue sections. The effect of Lon in glioma biology was investigated through siRNA targeting Lon. We also tested the in vitro antitumor activity of Lon inhibitor, CC4, in the glioma cell lines D-54 and U-251. High Lon expression was associated with high glioma tumor grade and poor patient survival. While Lon expression was elevated in response to a variety of stimuli, Lon knockdown in glioma cell lines decreased cell viability under normal conditions, and dramatically impaired glioma cell survival under hypoxic conditions. Furthermore, the Lon inhibitor, CC4, efficiently prohibited glioma cell proliferation and synergistically enhanced the therapeutic efficacy of the chemotherapeutic agents, temozolomide (TMZ) and cisplatin. We demonstrate that Lon plays a key role in glioma cell hypoxic survival and mitochondrial respiration, and propose Lon as a promising therapeutic target in the treatment of malignant gliomas.