Cholangiocarcinoma (CCA) is a malignant cancer with an unknown etiology and an unfavorable prognosis. Most patients are diagnosed at an advanced stage, thus making it essential to find novel curative targets for CCA. Metabolic reprogramming of the tumor cells includes metabolic abnormalities in glucose (known as the Warburg effect) and other substances such as amino acids and fats. Metabolic reprogramming produces anti-oxidant substances, reduces tumor oxidative stress, and finally promotes the proliferation of tumors. There is increasing evidence to imply that SIRT2, a histone deacetylase, and its downstream target cMYC, play metabolic regulatory roles in tumor cells. However, the role of the SIRT2/cMYC pathway in CCA is unclear. To assess the metabolic reprogramming function of the SIRT2/cMYC pathway in CCA and to determine the downstream targets as well as evaluate the therapeutic effect, the CCA RNA-Seq data were downloaded from the TCGA database. Differentially expressed genes were confirmed and KEGG pathway enrichment analysis was performed. Overall, 48 paired CCA samples were collected and subjected to immunohistochemical detection, and the clinical characteristics of participants were summarized. The CCA cells were suppressed or overexpressed with different downstream targets of SIRT2 and then subjected to apoptosis, immunoblotting, seahorse, and metabolites tracing analysis. In vivo experiments were also performed. We found that the SIRT2/cMYC pathway contributed to the proliferation of CCA cells and confirmed that the downstream target is PHDA1 and the serine synthesis pathway. The up-regulated SIRT2 and cMYC levels resulted in low levels of mitochondrial oxidative phosphorylation and increased conversion of glucose to serine and led to poor patient survival. The highly active SIRT2/cMYC pathway up-regulated the serine synthesis pathway pyruvate and increased antioxidant production, thus consequently protecting the CCA cells from oxidative stress-induced apoptosis. Our data revealed that the SIRT2/cMYC pathway plays a critical role in transforming glucose oxidative metabolism to serine anabolic metabolism, thus providing antioxidants for stress resistance. SIRT2/cMYC-induced metabolic reprogramming may represent a new therapeutic target for treating CCA. Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.