Macrolide–lincosamide–streptogramin B antibiotic resistance occurs through the action of erythromycin ribosome methylation (Erm) family proteins, causing problems due to their prevalence and high minimal inhibitory concentration, and feasibilities have been sought to develop inhibitors. Erms exhibit high conservation next to the N-terminal end region (NTER) as in ErmS, 64SQNF67. Side chains of homologous S, Q and F in ErmC’ are surface-exposed, located closely together and exhibit intrinsic flexibility; these residues form a motif X. In S64 mutations, S64G, S64A and S64C exhibited 71%, 21% and 20% activity compared to the wild-type, respectively, conferring cell resistance. However, mutants harboring larger side chains did not confer resistance and retain the methylation activity in vitro. All mutants of Q65, Q65N, Q65E, Q65R, and Q65H lost their methyl group transferring activity in vivo and in vitro. At position F67, a size reduction of side-chain (F67A) or a positive charge (F67H) greatly reduced the activity to about 4% whereas F67L with a small size reduction caused a moderate loss, more than half of the activity. The increased size by F67Y and F67W reduced the activity by about 75%. In addition to stabilization of the cofactor, these amino acids could interact with substrate RNA near the methylatable adenine presumably to be catalytically well oriented with the SAM (S-adenosyl-L-methionine). These amino acids together with the NTER beside them could serve as unique potential inhibitor development sites. This region constitutes a divergent element due to the NTER which has variable length and distinct amino acids context in each Erm. The NTER or part of it plays critical roles in selective recognition of substrate RNA by Erms and this presumed target site might assume distinct local structure by induced conformational change with binding to substrate RNA and SAM, and contribute to the specific recognition of substrate RNA.