We recently demonstrated that human aortic valve interstitial cells (HAVICs) obtained from patients with calcified aortic valve stenosis (AVS) were highly sensitive to ectopic calcification stimulation; however, the cell types contributing to calcification remains to be elucidated. We aimed to immunocytochemically characterize HAVICs and identify their contribution to valve calcification. HAVICs were isolated from patients with AVS and cultured on non-coated dishes. Immunocytochemical features and HAVIC differentiation were analyzed in passage 1 (P1). Most cultured P1 HAVICs were CD73-, CD90-, and CD105-positive, and CD45- and CD34-negative. HAVICs expressed vascular endothelial growth factor receptor 2 (VEGFR2); however, approximately 50% of the cells were α-smooth muscle actin (SMA)-positive, colonized, and easily differentiated into osteoblastic cells. Furthermore, VEGFR2-positive cells were more sensitive to tumor necrosis factor-α-induced ectopic calcification regardless of α-SMA expression. We concluded that HAVICs obtained from patients with calcified AVS are VEGFR2-positive undifferentiated mesenchymal cells and may contribute to aortic valve ectopic calcification.