Background: To investigate the correlation between gut microbiota and circulating microRNAs (miRNAs) in patients with primary diagnosis of type 2 diabetes mellitus (T2DM) and to explore the possible mechanisms of miRNA-gut microbiota crosstalke network in the regulation of the insulin signaling pathway and glucose homeostasis in T2DM., Methods: T2DM patients and normal controls were recruited. Fasting plasma and fecal samples were collected from the subjects, and their biochemical indexes including fasting blood glucose (FBG), glycated hemoglobin (HbAlc), cholesterol (TC), total triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and insulin were recorded. The variations in intestinal microbiota in the two groups were analyzed using 16S rRNA third-generation sequencing technology, and the differential expression of miRNAs between the groups was screened using miRNA high-throughput sequencing. The correlation and association between specifically changed intestinal microbiota and miRNA expressions were analyzed using a combination of bioinformatics analysis and statistical methods. Finally, 16S functional gene prediction analysis and target gene enrichment pathway analysis were carried out to predict relevant gut microbiota and miRNAs., Results: Compared with normal controls, the biochemical indexes of HAlbc, FBG, TG, TC, LDL, HDL, and insulin were significantly different in T2DM patients (P<0.001, P<0.001, P=0.0125, P=0.98, P<0.001 P=0.022, and P=0.0013, respectively). The two groups also showed significantly different intestinal microbiota distribution and miRNA expression characteristics, including in the counts of Bacteriodes. uniformis and Phascolarctobacterium. Faecium (P=0.023, 0.031), which were negatively correlated (P=0.014, FC = -2.36) with the expression levels of serum miR-122-5p (r=-0.68, -0.60, P=0.01, 0.01)., Conclusions: This study discovered specific gut microbiota and miRNA characteristics in patients with a primary diagnosis of T2DM. A negative correlation between miR-122-5p and the intestinal bacteria Bacteriodes. uniformis and Phascolarctobacterium. Faecium was also revealed, suggesting that the crosstalke between miRNA and gut microbiota may regulate the insulin secretion and signal transduction by controling key genes of glucose metabolism during the development of T2DM., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-6717). The authors have no conflicts of interest to declare., (2020 Annals of Translational Medicine. All rights reserved.)