Milking frequency influences milk production in cows by regulating milk synthesis and secretion. This in turn involves a number of genes that are differentially expressed. In this study, we hypothesized that milking frequency changes the milk yield through altered gene expression by modifying the methylation at CpGs dinucleotides located within regulatory regions. We focused on a candidate regulatory region of one casein gene (CSN1). Eight heifers were studied over three periods. Period 1: twice daily milking for 5 days of both udders of each heifer. Period 2: differential milking for 7 days: right and left udders were milked twice and once a day, respectively. Period 3: both udders returned to twice daily milking. Milk yield was recorded every day over the three periods. Biopsies from left & right udders were taken at the the end of period 2. Global DNA methylation was evaluated using the LUMA assay. DNA methylation of three CpGs located within a distal regulatory region CSN1 was analyzed using pyrosequencing after bisulfite treatment of genomic DNA. The mRNA expression levels have been quantified by RNASeq. Significance of results was studied with the Wilcoxon test. Global methylation of the once daily milking group (75.9%) is higher compared with the twice daily milking group (74.6%) but only significant if one of the eight heifers, which behaved differently from the others, was excluded (P=0.04). The DNA methylation of all three CpG in the once daily milking group is higher that that of twice daily milking group (P=0.02, P=0.07, and P=0.02, respectively). Once daily milking increases DNA methylation at a regulatory region of the CSN1 gene and at the global level in the bovine mammary gland, to a certain extent. These modifications are related to transcription of the casein genes and to some ncRNA.