DNA cytosine methylation occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring by DNA methyltransferases, resulting in 5-methylcytosine (5-mC). This process has been well studied and is generally associated with repression of gene expression. It was also observed that in humans, 5-mC occurs in various RNA molecules including tRNAs, rRNAs, mRNAs and non-coding RNAs (ncRNAs). At least 10,275 5-mC candidate sites were discovered in mRNAs and ncRNAs, which covered 10.6% of the total cytosine residues in the transcriptome. 5-mC seems to be enriched in some classes of ncRNA, but relatively depleted in mRNAs. However, the majority (83%) of their candidate sites were found in mRNAs. Within these transcripts 5-mC appears to be depleted within protein coding sequences but enriched in 5’ and 3’ UTRs. Two different methyltransferases, NSUN2 and Dnmt2 are known to catalyze the 5-mC modification in eukaryotic RNAs. Recent data strongly suggest that RNA cytosine methylation affects the regulation of various biological processes such as RNA stability and mRNA translation. Furthermore, loss of 5-mC in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of human disorders related to NSUN2-deficiency.