The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR knockout gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation.