Background: We explored the relationship between major electrocardiogram (ECG) abnormalities (mECG) and 25-hydroxy (25-OH) vitamin D deficiency (VDD) and the effect of mECG abnormalities on all-cause and cardiovascular mortality in a healthy cohort with 25-OH vitamin D insufficiency and deficiency. Hypothesis: Lower levels of serum 25-OH vitamin D are associated with increased prevalence of mECG on resting ECG. Methods: We identified 5108 individuals from the National Health and Nutrition Examination Survey-III. mECG abnormalities included: major Q-QS wave abnormalities, ST depression/elevation, negative T waves, Wolff-Parkinson-White pattern, and ventricular conduction defect. Our cohort was divided into 3 groups based on 25-OH vitamin D levels: Group 1 (referent): >40 ng/mL; group 2 (insufficient): ≥20.01 to ≤40 ng/mL; and group 3 (deficient): ≤20 ng/mL. Logistic regression and Cox proportional hazards regression models were built. Results: The prevalence of major ECG abnormalities across 25-OH vitamin D sufficiency, insufficiency, and deficiency was 5.9%, 11%, and 13 %, respectively (P = 0.01). VDD was an independent predictor of mECG abnormalities after adjusting for traditional risk factors (continuous variable odds ratio [OR]: 0.98, 95% confidence interval [CI]: 0.97-0.99, P = 0.007; categorical variable group 3 vs group 1 OR: 2.36, 95% CI: 1.1-5.12, P = 0.03). Baseline major ECG abnormalities were predictive of long-term all-cause (hazard ratio [HR]:1.52, 95% CI: 1.23-1.89), composite cardiovascular (HR: 1.7, 95% CI: 1.34-2.15), cardiovascular (HR: 1.64, 95% CI: 1.27-2.12), and ischemic heart disease mortality (HR: 1.98, 95% CI: 1.46-2.69) in individuals with 25-OH vitamin D levels ≤40 ng/mL. Conclusions: VDD is associated with increased prevalence of major ECG abnormalities. Well-structured trials are needed to assess progression/resolution of mECG abnormalities with vitamin D supplementation in deficient individuals.