Background: Nitric oxide (NO) exposure has been suggested to cause alterations in DNA methylation in breast cancer. We investigated the effect of NO on DNA methylation of promoters in cell lines of breast cancer. Material and Methods: The methylation status of the promoters of breast cancer 1 (BRCA1), deleted in colon cancer (DCC), Ras-association domain family 1A (RASSF1A), O-6-methylguanineDNA methyltransferase (MGMT), and secreted frizzled related protein 1 (SFRP1) were analyzed in the parental and high nitric oxide-adapted cell lines of breast cancer using Illumina MiSequencing. Results: Methylation of RASSF1A promoter in BT-20-HNO (74.7%) was significantly higher than that in BT-20 cells (72%) (p<0.05), whereas in MCF-7-HNO cells, methylation of MGMT promoter was found to have significantly decreased as compared to its parental cell line (45.1% versus 50.1%; p<0.0001). Promoter methylation of SFRP and DCC was elevated in T-47D-HNO relative to its parent cell line (p<0.05). Conclusion: Similarly to the double-edged effects of NO on tumorigenesis, its epigenetic effects through DNA methylation are diverse and contradictory in breast cancer.