Background <p>This study aimed to evaluate the prevalence of vitamin D deficiency and to assess the effects of vitamin D supplementation on myocardial and hepatic iron burden and organ function in children with transfusion-dependent beta-thalassemia (TDT).</p> Methods <p>A cross-sectional observational study was conducted in the pediatric hematology department of our institution. Patients aged three years and older with transfusion-dependent beta-thalassemia were included. Baseline and post-treatment laboratory assessments included serum calcium, phosphate, alkaline phosphatase, parathyroid hormone (PTH), and 25-hydroxyvitamin D [25(OH)D] levels. All patients received vitamin D supplementation according to their deficiency status. Cardiac and hepatic iron deposition were evaluated using T2* magnetic resonance imaging (MRI), and left ventricular ejection fraction (LVEF) was assessed by echocardiography.</p> Results <p>A total of 88 pediatric patients were enrolled. The largest proportion of patients was aged 4–9 years (<i>n</i> = 53), followed by those aged 10–14 years (<i>n</i> = 22) and 15–19 years (<i>n</i> = 13). The cohort consisted of 54.5% males (<i>n</i> = 48) and 45.5% females (<i>n</i> = 40). Patients were categorized into three groups based on serum 25(OH)D levels: severe deficiency (&lt; 10 ng/mL), mild deficiency (10–20 ng/mL), and sufficiency (≥ 20 ng/mL). Three months after vitamin D supplementation, statistically significant improvements were observed in both cardiac and hepatic MRI T2* values (<i>p</i> &lt; 0.05).</p> Conclusion <p>Vitamin D supplementation was associated with significant improvement in myocardial and hepatic iron parameters, suggesting a potential beneficial role in reducing iron-induced organ dysfunction. These findings highlight the importance of routine vitamin D monitoring and management in children with transfusion-dependent beta-thalassemia.</p>