<p>Prostate cancer (PCa) is among the most frequently occurring cancers in men. Its occurrence and progression are closely related to metabolic reprogramming, with abnormally active glycolysis being a notable feature sustaining rapid proliferation. Recent years have seen growing interest in the regulatory role of epigenetic modifications, particularly N⁶-methyladenosine (m⁶A) RNA methylation. AlkB homolog 5 (ALKBH5), as an m⁶A demethylase, may influence glycolysis in PCa by regulating target genes, offering new directions for mechanistic studies and therapy. The impact of different concentrations of Icariin (ICA) on PCa cell viability was first assessed using Cell Counting Kit-8 assays. Colony formation and Transwell assays were used to evaluate the effects of ICA on cell proliferation and migration, respectively. Apoptosis was detected by Annexin V-fluorescein isothiocyanate (FITC)/Propidium Iodide (PI) double staining. Glycolysis was evaluated by measuring the extracellular acidification rate (ECAR), oxygen consumption rate (OCR), and levels of glucose, lactate, and adenosine triphosphate (ATP). Methylated RNA immunoprecipitation-PCR was used to assess the effect of ICA on m⁶A methylation. The messenger RNA (mRNA) and protein expression of methyltransferase-like 3 (METTL3), METTL14, WTAP, fat mass and obesity-associated protein (FTO), and ALKBH5 were detected by quantitative real-time PCR and Western blot, respectively. Stable ALKBH5-knockdown and glutamyl-tRNA synthetase 2 (EARS2)-overexpressing PCa cell lines were constructed for functional studies. The half-life of EARS2 mRNA after ALKBH5 knockdown was determined using an actinomycin D assay to evaluate mRNA stability. RNA immunoprecipitation (RIP) experiments were performed to verify the interaction between ALKBH5 and EARS2 mRNA. In addition, a xenograft model was established by injecting PCa cells for in vivo validation. Immunohistochemistry (IHC) was used to detect protein expression in tumor tissues. ICA inhibited the proliferation (reduced cell viability, decreased colony formation), metastasis (impaired migration and invasion), and promoted apoptosis of PCa cells in a concentration-dependent manner, with no significant effect on cell viability at concentrations below 25 µM. ICA also concentration-dependently suppressed glycolysis in PCa cells. Treatment with 25 µM ICA reduced the global m⁶A methylation level and specifically upregulated the mRNA and protein expression of the demethylase ALKBH5, while the expression of other m⁶A-related regulatory factors was not significantly affected. Functional experiments showed that ALKBH5 knockdown or EARS2 overexpression promoted PCa cell proliferation, metastasis, and glycolysis (increased ECAR, glucose uptake, lactate production, ATP levels, and glycolysis-related protein expression; decreased OCR), and attenuated the inhibitory effects of ICA on these processes. RIP experiments confirmed that ALKBH5 interacts with EARS2 mRNA and that ALKBH5 reduces EARS2 mRNA stability via m⁶A demethylation, thereby downregulating its expression. In vivo, ICA effectively inhibited tumor growth in mice. IHC results showed that ICA upregulated ALKBH5 expression and downregulated the expression of hexokinase 2, lactate dehydrogenase A, and pyruvate kinase M2 in tumor tissues. These inhibitory effects of ICA were weakened by ALKBH5 knockdown. ICA suppresses the progression of PCa by inhibiting glycolysis, a process in which upregulation of ALKBH5 mediates m⁶A demethylation of EARS2 mRNA, leading to its destabilization and decreased expression. These findings were further validated in vivo.</p>