Recent studies have shown that DNA methylation in the promoter of microRNA-375 (miR-375) downregulates its expression during tumorigenesis. However, it is not known if CpG methylation of the miR-375 promoter also has a role in the pathogenesis of type 2 diabetes mellitus (T2DM). In this study, the expression level and CpG methylation status of miR-375 in patients with T2DM were analyzed. Plasma samples from 100 patients with T2DM and 100 healthy controls with normal glucose tolerance (NGT) were collected. The plasma levels of miR-375 were examined using quantitative polymerase chain reaction (qPCR) and the methylation status of 17 CpG sites in the promoter of the miR-375 were determined using MassARRAY spectrometry. The plasma levels of miR-375 were found to be upregulated in patients with T2DM compared with controls with NGT (P< 0.05). Overall, the methylation levels of the miR-375 promoter in patients with T2DM were not significantly different compared with controls with NGT; however, further studies revealed that four of the eight analyzed individual CpG units within the amplicon were significantly hypomethylated in T2DM samples compared with the NGT samples. This study demonstrated for the first time, to the best of our knowledge, that miR-375 is overexpressed in plasma in patients with T2DM, and this may be used as a novel biomarker to distinguish between patients with T2DM and healthy individuals. It was also demonstrated in this study that the miR-375 promoter is hypomethylated, in patients with T2DM, which may regulate the expression of miR-375 and contribute to the pathogenesis of T2DM.