Abstract Glioblastoma multiforme is the most common and aggressive primary brain tumor in adults. It is characterized by a rapid cell growth, CNS infiltration and is resistant to all known anticancer regimens. Therefore, development of effective therapies against these malignant brain tumors represents challenge for contemporary medicine. Anti-neoplastic potential of a common lipid-lowering drug, fenofibrate, is gaining a lot of attention, however, the precise mechanism of fenofibrate anti-cancer action remain to be elucidated. Fenofibric acid, which is an active metabolite of fenofibrate, is a potent agonist of peroxisome proliferator activated receptor alpha (PPARα), which transcriptional activity is expected to shift energy metabolism from glycolysis to fatty acid β-oxidation, therefore, it may be toxic to glycolytic tumor cells (Warburg effect). In parallel, to PPARα- dependent action, we have observed a strong PPARα- independent effect of fenofibrate on cancer cell energy metabolism, which was specific only to fenofibrate but not to fenofibric acid or to other PPARα agonists. We detected fenofibrate in mitochondrial membrane fractions of LN-229 cells treated with fenofibrate by using HPLC and subcellular fractionation. The accumulation of fenofibrate in mitochondrial membrane was accompanied by significant change in mitochondrial function, including loss of mitochondrial membrane potential, dramatic loss of oxygen consumption and lack of respiratory capacity measured as oxygen consumption rate (OCR). At the same time, fenofibrate increased the level of glycolysis (extracellular acidification rate, ECAR), possibly as compensation for impaired oxidative phosphorylation. In addition, fenofibrate-induced reduction of ATP levels triggered phosphorylation of AMPK and activation of autophagy. In conclusion, our results demonstrate two seemingly independent mechanisms of fenofibrate anticancer action: PPARα-dependent, which shifts cell metabolism from glycolysis to fatty acid β-oxidation and PPARα-independent, which causes accumulation of fenofibrate in cell membranes and inhibition of oxidative phosphorylation. Both mechanisms counteract to make this drug very effective against Glioblastoma tumors causing inhibition of cell growth, survival and finally leading to massive apoptosis of these practically incurable tumors. Citation Format: Anna M. Wilk, Adriana M. Zapata, Jennifer R. Mullinax, Dorota D. Wyczechowska, Krzysztof Reiss. PPAR alpha independent effect of fenofibrate on glioblastoma cancer metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5431. doi:10.1158/1538-7445.AM2013-5431 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.