5-Methylcytosine (m⁵C) is a well-characterized DNA modification, and is also predominantly reported in abundant non-coding RNAs in both prokaryotes and eukaryotes. However, the distribution and biological functions of m⁵C in plant mRNAs remain largely unknown. Here, we report transcriptome-wide profiling of RNA m⁵C in Arabidopsis thaliana by applying m⁵C RNA immunoprecipitation followed by a deep-sequencing approach (m⁵C-RIP-seq). LC-MS/MS and dot blot analyses reveal a dynamic pattern of m⁵C mRNA modification in various tissues and at different developmental stages. m⁵C-RIP-seq analysis identified 6045 m⁵C peaks in 4465 expressed genes in young seedlings. We found that m⁵C is enriched in coding sequences with two peaks located immediately after start codons and before stop codons, and is associated with mRNAs with low translation activity. We further demonstrated that an RNA (cytosine-5)-methyltransferase, tRNA-specific methyltransferase 4B (TRM4B), exhibits m⁵C RNA methyltransferase activity. Mutations in TRM4B display defects in root development and decreased m⁵C peaks. TRM4B affects the transcript levels of the genes involved in root development, which is positively correlated with their mRNA stability and m⁵C levels. Our results suggest that m⁵C in mRNA is a new epitranscriptome marker inArabidopsis, and that regulation of this modification is an integral part of gene regulatory networks underlying plant development.