RNA 5-methylcytosine (m C) is a prevalent RNA modification in multiple RNA species, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), and noncoding RNAs (ncRNAs), and broadly distributed from archaea, prokaryotes to eukaryotes. The multiple detecting techniques of m C have been developed, such as m C-RIP-seq, miCLIP-seq, AZA-IP-seq, RNA-BisSeq, TAWO-seq, and Nanopore sequencing. These high-throughput techniques, combined with corresponding analysis pipeline, provide a precise m C landscape contributing to the deciphering of its biological functions. The m C modification is distributed along with mRNA and enriched around 5'UTR and 3'UTR, and conserved in tRNAs and rRNAs. It is dynamically regulated by its related enzymes, including methyltransferases (NSUN, DNMT, and TRDMT family members), demethylases (TET families and ALKBH1), and binding proteins (ALYREF and YBX1). So far, accumulative studies have revealed that m C participates in a variety of RNA metabolism, including mRNA export, RNA stability, and translation. Depletion of m C modification in the organism could cause dysfunction of mitochondria, drawback of stress response, frustration of gametogenesis and embryogenesis, abnormality of neuro and brain development, and has been implicated in cell migration and tumorigenesis. In this review, we provide a comprehensive summary of dynamic regulatory elements of RNA m C, including methyltransferases (writers), demethylases (erasers), and binding proteins (readers). We also summarized the related detecting technologies and biological functions of the RNA 5-methylcytosine, and provided future perspectives in m C research. This article is categorized under: RNA Processing > RNA Editing and Modification.