Glucose metabolic reprogramming from oxidative phosphorylation to glycolysis is one of the hallmarks of cancer development. Coenzyme Q10 (CoQ10) is essential for electron transport in the mitochondrial respiratory chain and for antioxidant defense. Here, we investigated the role of a key factor in CoQ10 synthesis, prenyldiphosphate synthase subunit 2 (PDSS2), in hepatocellular carcinoma (HCC) tumorigenesis. PDSS2 was frequently downregulated in HCC tissues and was significantly associated with poorer HCC prognosis (P = 0.027). PDSS2 downregulation was a prognostic factor independent of T status and stage (P = 0.028). Downregulation of CoQ10 was significantly correlated with downregulation of PDSS2 in HCC tumor tissues (R = 0.414; P < 0.001). Of the six different splicing isoforms of PDSS2, the five variants other than full-length PDSS2 showed loss of function in HCC. Reintroduction of full-length PDSS2 into HCC cells increased CoQ10 and mitochondrial electron transport complex I activity and subsequently induced a metabolic shift from aerobic glycolysis to mitochondrial respiration in cells. Reintroduction of PDSS2 also inhibited foci formation, colony formation in soft agar, and tumor formation in nude mice. Knockdown of PDSS2 induced chromosomal instability in the MIHA immortalized human liver cell line. Furthermore, knockdown of PDSS2 in MIHA induced malignant transformation. Overall, our findings indicate that PDSS2 deficiency might be a novel driving factor in HCC development.Significance: Downregulation of PDSS2 is a driving factor in hepatocellular carcinoma tumorigenesis. Cancer Res; 78(16); 4471–81. ©2018 AACR.