Abstract: 5-methylcytosine (m⁵C) is an important post-translationally modified base in RNA formed by cytosine methylation. As the development of methylation sequencing techniques, extensive m⁵C modifications have been found in both coding and non-coding RNA. Incorporation of m⁵C into RNA is regulated by methyltransferases, demethylases, and m⁵C-binding proteins. m⁵C incorporation regulates RNA stability, transport, translation, and pressure stress. Recent studies have revealed that m⁵C incorporation into RNA is involved in the regulation of tumor pathogenesis, development, invasion, metastasis, and resistance. This article reviews m⁵C-mediated regulatory mechanisms and functions, and research progress on m⁵C-modified RNA functions in tumors, thus providing novel insights for developing innovative tumor diagnostic and therapeutic strategies.