Inappropriately high expression of matrix metalloproteinase 9 (MMP9) in the late stage of diabetic foot ulcers suppresses wound healing. The underlying mechanisms are not completely understood. Site-specific demethylation was reported to function in the regulation of genes, causing persistent high expression of target genes. Therefore, this study was designed to determine whether site-specific DNA demethylation was a key regulatory component of MMP9 expression in diabetic wound healing, and to further verify the crucial CpG site(s). Human keratinocyte cell line (HaCaT) cells were exposed to tumor necrosis factor a (TNFα), and changes in MMP9 expression and DNA methylation status were detected. We found TNFα treatment increased endogenous MMP9 expression in HaCaT cells and decreased the DNA methylation percentage at the -36 bp promoter site in a time-dependent manner. Bisulfite sequencing PCR revealed differentially demethylated CpG sites in the human MMP9 promoter region, but only the change at the -36 bp site was statistically significant. Dual-luciferase reporter assays showed that the promoter with only the -36 bp site demethylated had slightly higher transcriptional activity than the promoter with all other sites except the -36 bp site demethylated. Our results demonstrate that site-specific DNA demethylation plays an important role in MMP9 expression in TNFα-stimulated keratinocytes. The -36 bp site in the MMP9 gene promoter is crucial to this effect, but other CpG sites may exert synergistic effects. Collectively, these data may contribute to the future development of novel therapeutic strategies to treat diabetic foot ulcers and prevent gangrene and amputation.