HIF-1α has a broad impact on tumors, including enhanced utilization of glucose, tumor cell stemness, migration, metastasis and so on. In pilot study, we found that the expression of HIF-1α significantly increased in breast cancer cell lines and tissue samples with higher malignant behaviors and decreased in luminal subtype breast cancer cells and tissue samples. We analyzed and found there is one large CpG island in HIF-1α promoter around transcription start site, and the hypermethylation occurred at these CpGs and their surrounding non-CpGs sites. Epigenetic events driving tumorigenesis has been characterized. However, knowledge is lacking on the non-CpGs methylation of HIF-1α promoter in breast cancer cells. We validated that non-CpGs methylation can directly regulate HIF-1α expression by luciferase activity assay. We also found DNMT3a and Mecp2 play vital role in methylation at non-CpGs and CpGs sites. In addition, we noticed that H3K9ac modification could promote the transcription of HIF-1α in MDA-MB-231 cells by binding to the region contained hypomethylated non-CpG and CpG sites. Taken together, the hypomethylation status at non-CpG and CpG loci in HIF-1α promoter and H3K9ac modification together contribute to maintain higher HIF-1αactivity in invasive breast cancer cells when compared with the non-invasive breast cancer cells, which may establish a tissue-specific epigenetic modification pattern and point to the new directions for future understanding breast cancer therapy.