Calcification is an important pathological process and a common complication of degenerative valvular heart diseases, with higher incidence in aortic versus mitral valves. Two phenotypes of valvular interstitial cells (VICs), activated VICs and osteoblastic VICs (obVICs), synergistically orchestrate this pathology. It has been demonstrated that serotonin is involved in early stages of myxomatous mitral degeneration, whereas the role of serotonin in calcific aortic valve disease is still unknown. To uncover the link between serotonin and osteogenesis in heart valves, osteogenesis of aortic and mitral VICs was induced in vitro. Actin polymerization and serotonin signaling were inhibited using cytochalasin D and serotonin inhibitors, respectively, to investigate the role of cell activation and serotonin signals in valvular cell osteogenesis. To evaluate calcification progress, calcium and collagen deposits along with the expression of protein markers, including the rate-limiting enzyme of serotonin synthesis [tryptophan hydroxylase 1 (TPH1)], were assessed. When exposed to osteogenic culture conditions and grown on soft surfaces, passage zero aortic VICs increased extracellular collagen deposits and obVIC phenotype markers. A more intense osteogenic process was observed in aortic VICs of higher passages, where cells were activated prior to osteogenic induction. For both, TPH1 expression was upregulated as osteogenesis advanced. However, these osteogenic changes were reversed upon serotonin inhibition. This discovery provides a better understanding of signaling pathways regulating VIC phenotype transformation and explains different manifestations of degenerative pathologies. In addition, the discovery of serotonin-based inhibition of valvular calcification will contribute to the development of potential novel therapies for calcific valvular diseases.