Homocysteine (hcy) is an intermediate metabolite in the metabolic pathway of cysteine and methionine. As a non-coded amino acid, hcy is not normally incorporated into protein. However, homocysteine can be recognized and activated by methionyl-tRNA synthetase (MetRs) to produce Hcy-thiolactone (HTL), which can react with the ε-amino group of a protein lysine residue. The N-hcy-linked protein carrying a free thiol group can influence protein structure and function, thus leading to severe diseases. Histone has multiple specific dynamic post-translational modifications (PTMs), especially on the N-terminal tail of histones enriched with lysine and arginine residues. In this study, we confirmed that histone H3 can be modified by HTL on lysine residue. Relative and absolute quantification methods based on mass spectrometry demonstrated the crosstalk between methylation and acetylation of H3 in response to excess HTL. Overall, our data provide novel insights into histone modifications and the regulatory mechanisms of diseases related to homocysteinylation.