BACKGROUND: Intra-leaflet hemorrhage (IH) plays a well-recognized detrimental role in calcified aortic valve disease (CAVD). However, IH-induced fibro-osteogenic responses in valvular interstitial cells (VICs) appear to be triggered under specific pathological conditions. Iron deficiency (ID), a common co-morbidity in CAVD, may influence these responses. This study investigated the relationship between ID and pathological changes associated with CAVD, and its effects on IH-mediated fibro-osteogenic differentiation of VICs. METHODS AND RESULTS: Two independent studies were conducted, including 2495 patients in the discovery study and 34 in the validation study. Our data demonstrated that ID was associated with CAVD severity and progression, particularly in an age-dependent manner. Based on these clinical findings, immunofluorescence and Western blot analyses revealed that TFR1, a key iron import transporter, was significantly upregulated in human calcified aortic valves. Concurrently, iron accumulation was detected by Perl's staining in both calcific and non-calcific valve sections. In vitro , VICs cultured with human serum from ID patients showed red blood cell lysis-induced iron overload and fibro-calcific differentiation. CONCLUSIONS: ID triggers TFR1-mediated intracellular iron overload, leading to fibrosis and calcification in human VICs, thereby contributing to IH-mediated valve remodeling and calcification. These findings supported the potential role of monitoring and correcting ID to slow or prevent the progression of valvular calcification.