mRNA m5C, which has recently been implicated in the regulation of mRNA mobility, metabolism, and translation, plays important regulatory roles in various biological events. Two types of m5C sites are found in mRNAs. Type I m5C sites, which contain a 3’G-rich triplet motif and locate in the 5’ end of hairpin structures, are methylated by NSUN2. Type II m5C sites contain a 3’UCCA motif and locate in the loops of hairpin structures. However, their biogenesis remains unknown. Here we identified a novel mRNA methyltransferase that targets Type II m5C sites and generated BS-seq and RNA-seq data to verify our findings. Overall design: tRNA BS-seq was performed for HEK293T cell line, NSUN6 knockout HEK293T cell line, NSUN6 knockout HEK293T cell line rescued by different NSUN6 variants, wildtype flies and NSUN6 knockout flies.