Background: Rheumatic heart disease (RHD) imposes a significant burden on patients and the health system, especially in the developing countries. Recently, the role of vitamin D deficiency in increasing the severity of valve involvement and calcification in patients with RHD has been suggested. However, its role in the severe stenosis of the mitral valve is unknown. The aim of this study was to determine the relationship between serum levels of 25(OH)D and mitral involvement severity scores in patients with severe mitral stenosis (MS). Materials and Methods: In this cross-sectional study conducted in Shahid Madani Heart Center, Tabriz, Iran, 104 patients with severe rheumatic MS were reviewed, in the absence of other underlying diseases. Trans-esophageal Echocardiography was performed for all patients, and mitral valve Wilkins score and mitral valve area were calculated. Blood samples were taken from patients and levels of 25(OH)D, total calcium, phosphorus, and i-PTH were measured. Results: Eighty eight cases were female and 16 were male. Eighty-one patients had mild to moderate mitral calcification and in 23 patients with mitral valve was severely calcified. Furthermore, 54 were vitamin D deficient, while 35 had insufficient vitamin D levels, and only 15 had normal blood levels of vitamin D. In addition high serum iPTH levels were present in 83 patients (80%). The levels of 25(OH)D, calcium, phosphorous, and iPTH with total Wilkins score, calcification score and mitral valve area were not significantly correlated (p>0.05 for all above). Conclusions: Although no correlation was found between the severity of mitral valve involvement, and vitamin D and iPTH levels, vitamin D deficiency and secondary hyperparathyroidism tend to be common among patients with severe rheumatic MS, which may play a role in the progression of the disease.