METTL8 is a mitochondrial S-adenosyl-L-methionine-dependent methyltransferase that catalyzes N(3)-methylcytidine (m3C) modification at position 32 in the anticodon loop of mitochondrial tRNA(Ser)(UCN) and tRNA(Thr) 12. The enzyme localizes to the mitochondrial matrix through an N-terminal mitochondrial targeting sequence 32. Substrate recognition requires specific determinants: U34G35 motif and t6A37/(ms2)i6A37 modifications, which are present only in the two target mt-tRNAs 1. METTL8 also interacts with mitochondrial seryl-tRNA synthetase (SARS2), which modestly enhances its methylation activity 3. The m3C32 modification influences mt-tRNA structure and folding 21, and is essential for optimal mitochondrial protein translation and respiratory chain activity 45. Alternative splicing generates multiple METTL8 isoforms, with mitochondrial isoform 1 responsible for mt-tRNA modification and nucleolar isoform 4 potentially involved in R-loop regulation 3. METTL8 dysfunction impairs cortical neurogenesis by disrupting neural stem cell maintenance 4 and contributes to glioblastoma malignancy through HIF1α/RTK/Akt pathway activation 5. Additionally, METTL8 mediates lenvatinib resistance in hepatocellular carcinoma, making it a potential therapeutic target 6.