Background: Vitamin D (25(OH)D) deficiency has associations with bowl/colon cancer, arthritis, diabetes, and cardiovascular disease. Many athletes are vitamin D deficient, yet no studies have examined the association between 25(OH)D status and cardiac structure and function in healthy athletes. Design: A total of 506 national-level athletes [football (50%), handball (23%), volleyball (16%), and basketball (11%)] and 244 control participants presented for precompetition medical assessment. Controls were healthy individuals registered with a sporting federation undertaking <2 h of exercise per week. Methods: All individuals undertook a physical examination, 12-lead electrocardiogram, echocardiogram, and serum 25(OH)D evaluation. Results: From 506 athletes and 244 controls, 23 and 12.3% demonstrated 25(OH)D sufficiency (>30 ng/ml), 30 and 23.4% insufficiency (20-30 ng/ml), 37.2 and 48.8% deficiency (10-20 ng/ml), and 11 and 15.6% severe deficiency (<10 ng/ml). Severely 25(OH)D-deficient athletes present significantly (p<0.05) smaller aortic root and left atria diameters, intraventricular septum diameter (IVSd), left ventricular diameter during diastole (LVIDd), left ventricular mass (LVM), left ventricular volume during diastole (LVvolD), and right atrial (RA) area than insufficient and sufficient athletes. Furthermore, following logarithmic transformation adjusting 25(OH)D for age, body surface area, ethnicity, and athletic participation, positive associations were observed between 25(OH)D and IVSd, LVIDd, posterior wall thickness during diastole, LVM, and LVvolD in athletes but not in the control participants. Conclusions: Severely 25(OH)D-deficient athletes present significantly smaller cardiac structural parameters than insufficient and sufficient athletes. Future research should investigate the precise mechanism(s) causing cardiac hypertrophy with increases in serum 25(OH)D in healthy athletes.