Recently the effects of methionine (Met) supplementation in milk performance of dairy cows through gene expression regulation have become more evident. Histone methylation (HM) can affect gene expression and consequently milk biosynthesis. Therefore, we evaluated the effect of Met on histone methylation in bovine mammary epithelial alveolar cells (MacT) incubated at increased concentrations of l-methionine. Prior to transfection cells were cultivated in high glucose Dulbecco modified Eagle’s medium (DMEM) with sodium pyruvate and supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin and Fungizone antimycotic. The plasmids used in this study were the pcDNA3-K9 and pcDNA3-K27 (Addgene) for analysis of HM through fluorescence resonance energy transfer (FRET) technology. Cells were seeded 24 h before transfection at 30,000 cells/well in a 96-well plate. Cells were transfected with Lipofectamine 3000 as the transfection reagent at 0.3 uL/well and at 50 ng/well of plasmid in a reduced serum medium (OptiMEM) deprived of FBS. Transfected cells were treated for 24h in triplicates with 0, 125, 250, and 500 μMl-methionine. An inverted fluorescent microscope for live imagining (EVOS FL Auto) equipped with a motorized scanning stage, and an environment-controlled chamber at 37°C and 5.0% of CO₂ was used to take 4 pictures/well at 4x magnification 0, 12, and 24h post-treatment. Transfection efficiency, viability, and quantification of HM were assessed using the CellProfiler software. Data were analyzed using the PROC MIXED of SAS and significance was declared at P ≤ 0.05. Least squares means separation was corrected using Tukey’s test. Overall HM in K9 increased (P ≤ 0.05) in cells incubated with Met as early as 12h post-treatment, and this effect remained until 24h. The HM in K27 by Met seemed to be less effective, in fact, HM tended (P = 0.09) to be lower in cells incubated with 500 uM of Met than control. To expand on these effects, global DNA methylation and gene expression analysis will be performed. Our results indicate that Met treatment can affect the HM status of histone tail residues differently, and this can result in profound changes in gene expression regulation. The extent of dietary Met in HM at the mammary gland level and consequently milk synthesis remains unknown.