Altered microRNA (miRNA, miR) expression has been related to many disease processes; however, the miRNA expression signature in calcific aortic valve disease (CAVD) is unclear. In this study, microarrays were used to determine the miRNA expression signature of tissue samples from healthy individuals (n = 4) and patients with CAVD (n = 4). TargetScan, PITA, and microRNAorg 3-way databases were used to predict the potential target genes. DIANA-miRPath was used to incorporate the aberrant miRNAs into gene pathways. miRNA microarrays identified 92 differentially expressed miRNAs in CAVD tissues. The principal component analysis (PCA) of these samples and the unsupervised hierarchical clustering analysis based on the 92 aberrantly expressed miRNAs noted that miRNA expression could be categorized into two well-defined clusters that corresponded to healthy control and CAVD. Bioinformatic analysis showed the miRNA targets and potential molecular pathways. Collectively, our study reported the miRNA expression signature in CAVD and may provide potential therapeutic targets for CAVD.