Dietary choline can be a source of methyl groups for histone methylation (HM), which can affect gene expression and consequently milk biosynthesis. Therefore, we evaluated the effect of choline on histone methylation in bovine mammary epithelial alveolar cells (MacT). 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 24h before transfection at 30,000 cells/well in a 96-well plate. Cells were transfected with Lipofectamine 3000 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, 200, 400, and 800 ug/mL of choline. 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 and tendencies at P ≤ 0.15. Overall HM tend to decrease in K9 (P = 0.13) and increased in K27 (P ≤ 0.01) during the 24h treatment. In K9 transfected cells, the 400 and 800 choline treatments maintain a HM status, whereas HM decreased (P < 0.01) over time in the control and 200 choline. In K27 transfected cells, the 200 choline treatment produced the greatest (P < 0.01) HM by 24h post-treatment. To expand on these effects, global DNA methylation and gene expression analysis will be performed. Our results indicate that choline can affect the HM status of histone tail residues (K9 and K27) differently, and this may be reflected in transcriptional changes and consequently in milk biosynthesis.