Cardiovascular calcification is one of the most severe outcomes associated with cardiovascular disease and often results in significant morbidity and mortality. Previous reports indicated that epigenomic regulation of microRNAs (miRNAs) might play important roles in vascular smooth muscle cell (VSMC) calcification. Here, we identified potential key miRNAs involved in vascular calcification in vivo and investigated the role of miR-32-5p (miR-32). According to microarray analysis, we observed increased expression of miR-125b, miR-30a, and miR-32 and decreased expression of miR-29a, miR-210, and miR-320 during the progression of vascularcalcification. Additionally, gain- and loss-of-function studies of miR-32 confirmed promotion of VSMC calcification in mice through the enhanced expression of bonemorphogenetic protein-2, runt-related transcription factor-2(RUNX2), osteopontin, and the bone-specific phosphoprotein matrix GLA protein in vitro. Moreover, miR-32 modulated vascularcalcification progression by activating phosphoinositide 3-kinase (PI3K)signaling and increasing RUNX2 expression and phosphorylation by targeting the 3'-untranslated region of phosphatase and tensin homolog Mrna (PTEN) in mouse VSMCs. Furthermore, we detected higher miR-32 levels in plasmafrom patients with coronary artery disease with coronary artery calcification (CAC) as compared with levels observed in non-CAC patients (P = 0.016), further confirming miR-32 as a critical modulator and potential diagnostic marker for CAC.