Primary human aortic valvular interstitial cells (pHAVICs) play crucial roles in maintaining the mechanical structure and microenvironmental homeostasis of aortic valves. Pathologic processes such as inflammation, senescence, apoptosis, and metabolic disorders of valvular interstitial cells often lead to calcified aortic valve disease (CAVD). However, the lack of clinically relevant cellular models has impeded our understanding of CAVD. Here, we immortalized primary HAVICs with SV40 LTA. The iHAVICs (immortalized human aortic valvular interstitial cells) were maintained in a nonsenescent state and still had the potential to be induced into a senescent phenotype. In calcification induction experiments, iHAVICs can be induced to transform into osteogenic phenotypes via different stimuli via different pathways, accompanied by variations in different markers. In conclusion, we established and characterized a novel human immortalized aortic valve interstitial cell line as a practical in vitro experimental tool for the study of aortic valve calcification disease.