Simple Summary: Cancer stem cell have certain metabolic properties that are distinct from their differentiated counterparts. Our aim is to characterize CSC metabolism in oral cancer. Our study have several impacts: 1. Previous metabolic studies of CSC mainly focused on few energy metabolism pathway. Here we used novel transcription/metabolic joint analysis to reveal comprehensive metabolic alteration of CSC in oral cancer. 2. Assessing CSC metabolic phenotype in vivo is challenging. Here We used single-cell sequencing to explore the metabolic characteristics of CSC in vivo. 3. Our data suggested oral CSCs are metabolically inactive compared with differentiated cancer cells. This state may allow CSCs to resist the metabolic therapeutic strategies currently used for highly proliferative tumors. This knowledge may allow us to better develop metabolic therapy against CSC in oral cancer. Understanding the distinct metabolic characteristics of cancer stem cells (CSC) may allow us to better cope with the clinical challenges associated with them. In this study, OSCC cell lines (CAL27 and HSC3) and multicellular tumor spheroid (MCTS) models were used to generate CSC-like cells. Quasi-targeted metabolomics and RNA sequencing were used to explore altered metabolites and metabolism-related genes. Pathview was used to display the metabolites and transcriptome data in a KEGG pathway. The single-cell RNA sequencing data of six patients with oral cancer were analyzed to characterize in vivo CSC metabolism. The results showed that 19 metabolites (phosphoethanolamine, carbamoylphosphate, etc.) were upregulated and 109 metabolites (2-aminooctanoic acid, 7-ketocholesterol, etc.) were downregulated in both MCTS cells. Integration pathway analysis revealed altered activity in energy production (glycolysis, citric cycle, fatty acid oxidation), macromolecular synthesis (purine/pyrimidine metabolism, glycerophospholipids metabolism) and redox control (glutathione metabolism). Single-cell RNA sequencing analysis confirmed altered glycolysis, glutathione and glycerophospholipid metabolism in in vivo CSC. We concluded that CSCs are metabolically inactive compared with differentiated cancer cells. Thus, oral CSCs may resist current metabolic-related drugs. Our result may be helpful in developing better therapeutic strategies against CSC. [ABSTRACT FROM AUTHOR]