Objective · To investigate the effect of histone H3K14M mutation on the activity of methyltransferase RomA, a secreted effector of Legionella pneumophila in eukaryotic cells and the underlying mechanisms. Methods · Wide-type histone H3 (H3WT) and mutant histone H3 (the lysine residue 14 was replacedmethionine, isoleucine or arginine residue, and named as H3K14M, H3K14I, and H3K14R, respectively) recombinant plasmids were constructed. Packaged lentiviruses with these plasmids were used to infect eukaryotic cells 293T and THP-1 with or without over- of RomA. The H3K14 methylation and acetylation were analyzedWestern blotting. The interaction of RomA with H3WT and H3K14 mutants was detectedco-immunoprecipitation. Results · A secreted effector of Legionella pneumophila named RomA targeted the host cell nucleus to upregulate the H3K14 methylation level and downregulate the H3K14 acetylation level for inhibiting the gene in host cells and promoting Legionella pneumophila’s efficient intracellular replication. But histone H3K14M mutation could promote the interaction between H3K14M and RomA and thus inhibited the methyltransferase activity of RomA. Conclusion · Histone H3K14M mutation significantly inhibits the activity of Legionella pneumophila methyltransferase RomA.