〔Abstract〕 Objective To analyze the DNA methylation spectrum of mouse islet β cell line(MIN6) using MassARRAY platform, and to explore the changes of methylation model by treating MIN6 cells with DNA demethylation drug 5-aza-2′-deoxycytidine(5-Aza-CdR). Methods The methylation status of miR-375 gene DNA in MIN6 cell line and mouse embryonic fibroblast cell line(NIH3 T3) were detected by MassARRAY platform. The changes of miR-375 gene methylation status were evaluated after treatment with different concentrations of 5-Aza-CdR. The effect of 5-Aza-CdR on the growth and proliferation of MIN6 cells was detected by Thiazolyl blue tetrazolium bromide(MTT). The expression of miR-375 in MIN6 cells and untreated NIH3 T3 cells was detected by qRT-PCR. Results 5-Aza-CdR could inhibit the growth of MIN6 cells. After treated with 5-Aza-CdR for 24,48,72 h, the OD value of 2 μmol/L concentration group was different from that of other groups(P<0.05), and the difference was statistically significant at different time points(P<0.05). After MIN6 cells were treated with different concentrations of 5-Aza-CdR for 24,48,72 h, the DNA of mmu-miR-375 in each group was highly methylated, and the average methylation rate of miR-375 DNA in 50 μmol/L group was lower than that in other groups. The expression of mmu-miR-375 in MIN6 cells treated with 5-Aza-CdR in 50 μmol/L group was significantly higher than that in 0 μmol/L group after 24,48,72 h(P<0.05). The DNA expression of mmu-miR-375 gene in MIN6 cells was significantly lower than that in NIH3 T3 cells(P<0.05). Conclusion These results indicate that promoter hypermethylation of the mmu-miR-375 is a common event in MIN6 cells.