Background <p>Diabetic kidney disease (DKD) is a serious microvascular complication of type 2 diabetes mellitus (T2DM). miR-127-3p is dysregulated in T2DM, but the specific molecular mechanism remains unclear. We aim to probe the diagnostic value of miR-127-3p and its molecular mechanism in T2DM and DKD.</p> Methods <p>This study comprised 218 individuals, including 78 patients with T2DM, 72 patients with DKD and 68 healthy controls. All participants underwent fasting peripheral blood collection. In vitro, we simulated a hyperglycemic environment by treating human mesangial cells (HMC) with high-concentration glucose (HG). Subsequently, RT-qPCR was used to detect the levels of miR-127-3p in serum and HMC. Cell viability and inflammatory cytokine (TNF-α, IL-1β and IL-6) levels were assessed using the CCK-8 assay and ELISA, respectively. The dual-luciferase reporter assay validated the target relationship between miR-127-3p and ACO2.</p> Results <p>By comparing baseline clinical characteristics, we identified significant differences among the three groups in high density lipoprotein cholesterol (HDL-C), triglycerides (TG), fasting blood glucose (FBG), glycated hemoglobin A1c (HbA1c), blood urea nitrogen (BUN), estimated glomerular filtration rate (eGFR) and albuminuria. Additionally, miR-127-3p was elevated in T2DM and DKD patients. It could distinguish healthy individuals from T2DM or T2DM from DKD. In HG-induced HMC, miR-127-3p inhibitor elevated the cell viability and the levels of SOD while suppressing the levels of MDA. These effects were abolished by ACO2 silencing. Furthermore, downregulated miR-127-3p reduced the levels of TNF-α, IL-1β and IL-6. sh-ACO2 alleviated the inhibitory effects of miR-127-3p.</p> Conclusions <p>Upregulated miR-127-3p was involved in the progression of T2DM and DKD. In HG-induced HMC, down-regulated miR-127-3p improved cell viability and suppressed oxidative stress and inflammatory responses by negatively regulating ACO2.</p>