Deubiquitylating enzymes (DUBs) play an essential role in targeted protein degradation and represent an emerging therapeutic paradigm in cancer. However, their therapeutic potential in cholangiocarcinoma (CCA) has not been explored. Herein, based on The Cancer Genome Atlas (TCGA) and The Gene Expression Omnibus (GEO) databases, we found that ubiquitin-specific protease 21 (USP21) was upregulated in CCA, high USP21 level was associated with poor prognosis. In vivo and in vitro, we identified USP21 as a master regulator of CCA growth and maintenance, which directly interacted with deubiquitinates and stabilized the heat shock protein 90 (HSP90) through K48-linked deubiquitination, and in turn, this stabilization increased HIF1A expression, thus upregulating key glycolytic enzyme genes ENO2, ENO3, ALDOC, ACSS2, and then promoted aerobic glycolysis, which provided energy for CCA cell proliferation. In addition, USP21 could directly stabilize alpha-Enolase 1 (ENO1) to promote aerobic glycolysis. Furthermore, increased USP21 level enhanced chemotherapy resistance to the gemcitabine-based regimen. Taken together, we identify a USP21-regulated aerobic glycolysis mechanism that involves the USP21/HSP90/HIF1A axis and USP21/ENO1 axis in CCA tumorigenesis, which could serve as a potential target for the treatment of CCA.