Prolactin stimulates the expression of milk genes during lactation through the activation of STAT5 transcription factors, which subsequently bind to their cognate target sequence on the promoters. Demethylation of 5methylCpG dinucleotides permits the tissue-specific accessibility of transcription factor-binding sites during development, but remethylation has not been shown to contribute to acute suppression of gene expression. We characterize functionally a novel STAT5-binding lactational enhancer in the far upstream promoter (approximately -10 kbp) of the bovine alphaS1-casein-encoding gene. This promoter area is hypo-methylated in the lactating udder only. Remethylation of this area accompanies an experimentally elicited acute shutdown of casein synthesis in fully lactating cows, whose udder quarters have experimentally been infected with a pathogenic E. coli strain. Within 24 h after infection, the relevant promoter area was remethylated from 10% of the DNA molecules in the uninfected control quarters to approximately 50% in the infected quarters, the typical values for fully lactating and not lactating udders respectively. Increased methylation resulted in tighter chromatin packing. Concomitantly, the alphaS1-casein mRNA concentration dropped to approximately 50% while the protein synthesis was shut down to approximately 2.5% in the infected quarters, alone. The methylation status of the promoter from a not lactationally regulated gene was unaltered, and the distal alphaS1-casein promoter was not remethylated in udder quarters with subclinical Staphylococcus aureus infections featuring sustained casein synthesis. Hence, infection-related remethylation of the alphaS1-casein promoter and chromatin remodelling serves as an acute, spatially restricted regulatory mechanism, which might insulate the promoter against the systemically unchanged high levels of circulating prolactin. This provides a rare example for an acute regulatory significance of CpG methylation.