INTRODUCTION AND OBJECTIVES: Deregulation of m MATERIALS AND METHODS: Expression analysis was done by quantitative PCR, immunoblotting, or immunohistochemistry. Cell sphere formation, invasiveness, apoptosis, proliferation and viability were detected by sphere formation, transwell, flow cytometry, EdU and CCK-8 assays, respectively. Xenograft studies were performed to analyze the role in vivo. Methylated RNA immunoprecipitation (MeRIP) and RIP assays were used to verify the METTL16/PFKM relationship. PFKM mRNA stability was tested by actinomycin D treatment. Chromatin immunoprecipitation (ChIP) and luciferase assays were performed to analyze the POU3F2/METTL16 relationship. RESULTS: In HCC, METTL16 expression was elevated, and increased levels of METTL16 transcript predicted poor HCC prognosis. METTL16 deficiency resulted in suppressed HCC cell growth, invasiveness and sphere formation. Moreover, METTL16 depletion diminished HCC cell glycolysis. Mechanistically, PFKM expression was positively associated with METTL16 expression. METTL16 mediated m6A methylation to stabilize PFKM mRNA via an IGF2BP3-dependent manner. Restored PFKM expression exerted a counteracting effect on METTL16 deficiency-mediated in vitro cell phenotype alterations and in vivo xenograft growth suppression. Furthermore, POU3F2 promoted the transcription of METTL16 in HCC cells. CONCLUSIONS: Our findings define the crucial role of the POU3F2/METTL16/PFKM axis in HCC pathogenesis, offering the potential opportunity to combat HCC.