5-methylcytosine (m5C) plays a crucial role in gene expression regulation and RNA metabolism, and it is an important form of RNA epigenetic modification. This article systematically summarizes the molecular mechanisms of m5C regulation, including its dynamic regulatory network composed of methyltransferases, demethylases, and recognition proteins, and explores the function and mechanism of m5C in RNA stability, translation, and nucleocytoplasmic transport. m5C participates in the development of tumors by regulating oncogene expression, immune microenvironment remodeling, and metabolic reprogramming. The correlation between m5C modification levels and tumor patient prognosis is analyzed, and the potential value of m5C as a tumor diagnostic biomarker and therapeutic target is explored. High throughput sequencing and computational biology have driven m5C investigation, but the development and innovation of detection technologies, mechanisms for regulating tissue heterogeneity, and synergistic effects with other modifications such as m6A remain current challenges. In future, it needs to further be analyzed the precise regulatory network of m5C, providing theoretical basis for developing targeted intervention strategies to promote its application in precision medicine.