Shift work, particularly, night work has been classified as a probable carcinogen to humans based on the increased risk observed in epidemiological studies for some cancer types, including female breast cancer. The underlying molecular mechanisms are not well established, but may involve aberrant epigenetic modifications. Here, effects of changes in methylation status of 5-methyl cytosine in melatonin and female hormone receptor genes were investigated as possible mechanisms for increased breast cancer risk in female night shift workers. Methylation in promoter regions of the MTNR1A, MTNR1B, PGR, ESR1 and ESR2 genes was analyzed by pyrosequencing in a nested case-control study of female nurses, including 354 breast cancer cases and 356 healthy controls. The effects of methylation as well as the combined effects of methylation and shift work on breast cancer risk were assessed. We demonstrate that increased methylation of the MTNR1A promoter is associated with increased risk of breast cancer (OR=1.13, 95% CI: 1.02 -1.24, P=0.019). No association between promoter methylation levels and breast cancer risk was observed for the other receptor genes investigated. Furthermore, MTNR1A methylation levels were not affected by shift work. Altogether, our data suggest that epigenetic regulation of MTNR1A may contribute to breast cancer.