The role of microRNAs (miRNAs) in vascular calcification is currently unclear. To examine how miRNAs are involved in vascular smooth muscle cell (VSMC) calcification, we explored the alteration of miRNAs in VSMC calcification in vitro and in vivo. Klotho homozygous mutant mice (kl/kl) display vascular calcification and have perturbations of calcium handling. We therefore hypothesized that the calcium perturbations in VSMCs could be mediated by miRNAs. Using an miRNA array analysis, we demonstrated that miRNAs are aberrantly expressed in the aortic media of 3-week-old kl/kl mice compared with wild-type (WT) mice. The expression levels of miR-135a(*), miR-762, miR-714, and miR-712(*) in the aortic media of kl/kl mice were significantly higher than in WT mice. We used quantitative real-time reverse transcriptase polymerase chain reaction to further confirm that these miRNAs were increased in the aortic media of kl/kl mice and in cultured VSMCs treated with high phosphate and calcium. A search of the miRNA database indicated that the Ca(2+) efflux proteins NCX1, PMCA1, and NCKX4 frequently appeared as potential targets of these miRNAs. The transfection of miRNA mimics into cultured VSMCs reduced the protein levels of each potential target. Conversely, miRNA inhibitors reduced phosphate and calcium-induced VSMC calcification. Furthermore, these inhibitors decreased the intracellular Ca(2+) concentration in cultured VSMCs after treatment with phosphate and calcium. Our results suggest that increased expression of miR-135a(*), miR-762, miR-714, and miR-712(*) in VSMCs may be involved in VSMC calcification by disrupting Ca(2+) efflux proteins.