Mitochondrial dysfunction and glycolysis activation are improtant hallmarks of hepatocellular carcinoma (HCC). NOP2 is an S-adenosyl-L-methionine-dependent methyltransferase that regulates the cell cycle and proliferation activities. In this study, found that NOP2 contributes to HCC progression by promoting aerobic glycolysis. Our results revealed that NOP2 was highly expressed in HCC and that it was associated with unfavorable prognosis. NOP2 knockout in combination with sorafenib enhanced sorafenib sensitivity, which, in turn, led to marked tumor growth inhibition. Mechanistically, we identified that NOP2 regulates the c-Myc expression in an m5C-modification manner to promote glycolysis. Moreover, our results revealed that m5C methylation induced c-Myc mRNA degradation in an eukaryotic translation initiation factor 3 subunit A (EIF3A)-dependent manner. In addition, NOP2 was found to increase the expression of the glycolytic genes LDHA, TPI1, PKM2, and ENO1. Furthermore, MYC associated zinc finger protein (MAZ) was identified as the major transcription factor that directly controlled the expression of NOP2 in HCC. Notably, in a patient-derived tumor xenograft (PDX) model, adenovirus-mediated knockout of NOP2 maximized the antitumor effect and prolonged the survival of PDX-bearing mice. Our cumulative findings revealed the novel signaling pathway MAZ/NOP2/c-Myc in HCC and uncovered the important roles of NOP2 and m5C modifications in metabolic reprogramming. Therefore, targeting the MAZ/NOP2/c-Myc signaling pathway is suggested to be a potential therapeutic strategy for the treatment of HCC.