BACKGROUND: Protein arginine methylations are crucial post-translational modifications (PTMs) in eukaryotes, playing a significant regulatory role in diverse biological processes. Here, we present our investigation into the detailed arginine methylation pattern of the C-terminal RG-rich region of METTL14, a key component of the m6A RNA methylation machinery, and its functional implications in biology and disease. METHODS: Using ETD-based mass spectrometry and in vitro enzyme reactions, we uncover a specific arginine methylation pattern on METTL14. RNA methyltransferase activity assays were used to assess the impact of sDMA on METTL3:METTL14 complex activity. RNA immunoprecipitation was used to evaluate mRNA-m6A reader interactions. MeRIP-seq analysis was used to study the genome-wide effect of METTL14 sDMA on m6A modification in acute myeloid leukemia cells. RESULTS: We demonstrate that PRMT5 catalyzes the site-specific symmetric dimethylation at R425 and R445 within the extensively methylated RGG/RG motifs of METTL14. We show a positive regulatory role of symmetric dimethylarginines (sDMA) in the catalytic efficiency of the METTL3:METTL14 complex and m6A-specific gene expression in HEK293T and acute myeloid leukemia cells, potentially through the action of m6A reader protein YTHDF1. In addition, the combined inhibition of METTL3 and PRMT5 further reduces the expression of several m6A substrate genes essential for AML proliferation, suggesting a potential therapeutic strategy for AML treatment. CONCLUSIONS: The study confirms the coexistence of sDMA and aDMA modifications on METTL14's RGG/RG motifs, with sDMA at R425 and R445 enhancing METTL3:METTL14's catalytic efficacy and regulating gene expression through m6A deposition in cancer cells.