Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in mRNA and exemplified by the critical roles of RNA modifications in normal physiology and disease. Despite a resurgent interest in these modifications, the biochemistry of 3-methylcytidine (m 3 C) formation in mammalian RNAs is still poorly understood. However, the recent discovery of  trm141  as the second gene responsible for m 3 C presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in m 3 C formation in mammals as well. Here, we report the discovery and characterization of three distinct m 3 C-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute m 3 C in specific tRNAs and that METTL8 only contributes m 3 C to mRNA. MS analysis revealed that there is an ∼30-40% and ∼10-15% reduction, respectively, in  METTL2  and  -6  null-mutant cells, of m 3 C in total tRNA, and primer extension analysis located METTL2-modified m 3 C at position 32 of tRNA Thr  isoacceptors and tRNA Arg(CCU)  We also noted that METTL6 interacts with seryl-tRNA synthetase in an RNA-dependent manner, suggesting a role for METTL6 in modifying serine tRNA isoacceptors.  METTL8 , however, modified only mRNA, as determined by biochemical and genetic analyses in  Mettl8  null-mutant mice and two human  METTL8  mutant cell lines. Our findings provide the first evidence of the existence of m 3 C modification in mRNA, and the discovery of METTL8 as an mRNA m 3 C writer enzyme opens the door to future studies of other m 3 C epitranscriptomic reader and eraser functions.