Objective: This study aims to explore the relationship between the methylation of matrix metalloproteinase (MMP)-9 gene promoter region and diabetic nephropathy (DN) through the detection of the methylation level of MMP-9 gene promoter region in the peripheral blood of patients with DN in different periods and serum MMP-9 concentration. Methods: The methylation level of the MMP-9 gene promoter region was detected by methylation-specific polymerase chain reaction (MSP), and the content of MMP-9 in serum was determined by enzyme-linked immunosorbent assay (ELISA). Results: Results of the statistical analysis revealed that serum MMP-9 protein expression levels gradually increased in patients in the simple diabetic group, early diabetic nephropathy group and clinical diabetic nephropathy group, compared with the control group; and the difference was statistically significant (P < 0.05). Compared with the control group, the methylation levels of MMP-9 gene promoter regions gradually decreased in patients in the simple diabetic group, early diabetic nephropathy group, and clinical diabetic nephropathy group; and the difference was statistically significant (P < 0.05). Furthermore, correlation analysis results indicated that the demethylation levels of the MMP-9 gene promoter region was positively correlated with serum protein levels, urinary albumin to creatinine ratio (UACR), urea and creatinine; and was negatively correlated with GFR. Conclusion: The demethylation of the MMP-9 gene promoter region may be involved in the occurrence and development of diabetic nephropathy by regulating the expression of MMP-9 protein in serum.