Objectives: Milk production in cows depends on all milk component synthesis and secretion by the mammary gland (protein, sugar, lipids…). This in turn involves a number of genes that are differentially expressed in this organ. In this study, we hypothesized that changes in gene expression profiles are related to differences in the methylation level at CpGs dinucleotides located within distal regulatory regions. As a first step, we focused on candidate regulatory regions of one casein gene (CSN1S1). Experimental methods: Holstein heifers were studied during early pregnancy (4 cows, day 60 of pregnancy) and lactation (8 cows, 7-10 weeks post-delivery). During lactation, the cows 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 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 and ncRNA expression levels have been quantified by RNASeq. Significance of results was studied with the Wilcoxon test. All experiments were conducted in compliance with French regulations on animal experimentation and with the authorization of the French ministry of Agriculture (Animal health and protection directorate, accreditation number 78-21). Results: During lactation when cows are milked twice daily, global DNA methylation in the mammary gland reaches 74.1%. The DNA methylation of each three CpG of a distal regulatory region of the CSN1S1 gene is low (33.1, 34.2 and 27.7%, respectively). In most cases (60%), the three CpG are simultaneously non methylated whereas only 17.8% are simultaneously methylated during lactation. In the once daily milking group, global DNA methylation is higher (75.9%) compared to the twice daily milking group but only significant if one of the eight heifers, which behaved differently from the others, was excluded (P=0.04). In the once daily milking group, the methylation level of each CpG is higher than that of twice daily milking group (P=0.02, P=0.07, and P=0.02, respectively). Furthermore, once daily milking induces inverse shifts with a decrease in simultaneaous non methylation (48%) and an increase in simultaneous methylation of the three CpG (26%). During pregnancy, the methylation level of each of these CpG is higher (57.1, 59.5, 50.1%, respectively). RNAseq data obtained from the mammary gland of those cows reveal the expression of ncRNA, specific to the mammary gland, in the vicinity of this region. Conclusion: A distal regulatory region of the CSN1S1 gene is hypomethylated during lactation as compared to pregnancy. Its methylation level increases during once daily milking and to a certain extent this corresponds to an increase in global DNA methylation state in the bovine mammary gland. These modifications may be related to transcription of the casein genes and to some ncRNA.