3-Methylcytidine (m3C), a prevalent modification of transfer RNAs (tRNAs), was recently identified in eukaryotic messenger RNAs (mRNAs). However, its precise distribution and formation mechanisms in mRNAs remain elusive. Here, we develop a novel approach, m3C immunoprecipitation and sequencing (m3C-IP-seq), utilizing antibody enrichment to profile the m3C methylome at single-nucleotide resolution. m3C-IP-seq captures 12 cytoplasmic tRNA isoacceptors and 2 mitochondrial tRNA isoacceptors containing m3C modifications. Moreover, m3C-IP-seq permits the comprehensive profiling of m3C sites in mRNAs and long noncoding RNAs, with their presence reliant on a nuclear isoform of METTL8. A significant proportion of m3C sites is concentrated in the 3' untranslated region (3' UTR) of mRNAs and is associated with mRNA degradation. Additionally, m3C methylation is dynamic and responds to hypoxia. Collectively, our data demonstrate the widespread presence of m3C modification in the human transcriptome and provide a resource for functional studies of m3C-mediated RNA metabolism.