Objective To investigate the regulating effects and mechanism of microRNA (miRNA)-141 on bone morphogenetic protein-2 (BMP-2)-induced calcification of human aortic valve. Methods Twenty-four samples of human degenerative aortic valve were collected, and the mRNA and protein expression levels of miRNA-141 and BMP-2 were detected by quantitative real-time polymerase chain reaction and Western blotting. miRNA-141 was up/down-regulated in human aortic valve interstitial cells (HAVICs), Von Kossa staining was used to show cellular calcification, and mRNA expression of distal-less homeobox 5 (Dlx5) and protein expression of BMP-2 were compared. Dual luciferase experiment was used to verify whether Dlx5 was the target gene of miRNA-141. miRNA-141 was up/down-regulated in aortic valve calcification mouse models with or without Dlx5 knockout, Von Kossa staining was used to compare aortic valve calcification, and BMP-2 protein expression was detected. Results Compared with normal aortic valve tissues, the expression of miRNA-141 was significantly decreased in degenerative aortic valves (1.00±0.02 vs 0.35±0.06, P=0.01), while the mRNA and protein expression levels of BMP-2 were significantly increased (both P=0.01). In HAVICs, the up/down regulation of miRNA-141 could inhibit/promote calcification (P=0.02 or P=0.01), and decrease/increase the mRNA expression of Dlx5 (both P=0.01) and the protein expression of BMP-2 (P=0.02 or P=0.01). Dual luciferase experiment validated that miRNA-141 directly targeted Dlx5. In aortic valve calcification mouse model, up/down-regulation of miRNA-141 could inhibit/promote calcification (both P<0.05), and decrease/increase the mRNA and protein expression levels of Dlx5 and BMP-2 (all P<0.05); while in mouse model with Dlx5 knockout, there were no correlation between miRNA-141 expression and valvular calcification or BMP-2 expression. Conclusion miRNA-141 can inhibit human aortic valve calcification via regulating BMP-2 by targeting Dlx5.