--- - |2 Abstract Vascular calcification is highly prevalent in patients with type 2 diabetes mellitus (T2DM), one of the most common chronic diseases with high morbidity and mortality. In recent years, microRNAs have been widely reported as potential biomarkers for the diagnosis and treatment of T2DM. We hypothesized that miR‐128‐3p is associated with cardiovascular calcification and insulin resistance (IR) in rats with T2DM by targeting ISL1 via the Wnt pathway. Microarray analysis was adopted to identify differentially expressed genes related to T2DM. T2DM models were induced in rats. Blood samples from normal and T2DM rats were used to detect islet β‐cell function, islet sensitivity, and calcium content. Next, islet tissues were obtained to identify the expression of miR‐128‐3p, ISL1, and the Wnt signaling pathway‐ and apoptosis‐related genes. Finally, apoptosis of islet β‐cells was determined by flow cytometry. Through microarray analysis of GSE27382 and GSE23343, ISL1 was found to be downregulated in T2DM. In blood samples from T2DM rats, basic biochemical indicators, IR, and calcium content were increased, and islet sensitivity and islet β‐cell function were decreased. Furthermore, upregulation of miR‐128‐3p and ISL1 gene silencing promoted the expression of Wnt‐1, β‐catenin, GSK‐3β, and Bax and the phosphorylation of β‐catenin and GSK‐3β, inhibited c‐fos, PDX‐1, and Bcl‐2 expression, and enhanced cell apoptosis. The key findings of our study demonstrate that miR‐128‐3p aggravates cardiovascular calcification and IR in T2DM rats by downregulating ISL1 through the activation of the Wnt pathway. Thus, miR‐128‐3p may serve as a potential target for the treatment of T2DM. - Journal of Cellular Physiology, EarlyView.