Objective:Leaflet thickening, fibrosis, and hardening are early pathological features of calcific aortic valve disease (CAVD). An inadequate understanding of the resident aortic valve cells involved in the pathological process may compromise the development of therapeutic strategies. We aim to construct a pattern of the human aortic valve cell atlas in healthy and CAVD clinical specimens, providing insight into the cellular origins of CAVD and the complex cytopathological differentiation process.Approach and Results:We used unbiased single-cell RNA sequencing for the high-throughput evaluation of cell heterogeneity in 34 632 cells isolated from 6 different human aortic valve leaflets. Cellular experiments, in situ localization, and bulk sequencing were performed to verify the differences between normal, healthy valves and those with CAVD. By comparing healthy and CAVD specimens, we identified 14 cell subtypes, including 3 heterogeneous subpopulations of resident valve interstitial cells, 3 types of immune-derived cells, 2 types of valve endothelial cells, and 6 novel valve-derived stromal cells found particularly in CAVD leaflets. Combining additional verification experiments with single-cell transcriptome profiling provided evidence of endothelial to mesenchymal transition involved in lesion thickening of the aortic valve leaflet.Conclusions:Our findings deconstructed the aortic valve cell atlas and suggested novel functional interactions among resident cell subpopulations. Our findings may provide insight into future targeted therapies to prevent CAVD.