Background: Atrial fibrillation (AF) is the most common arrhythmia worldwide and a large proportion of patients with AF are older than 75 years of age. Mitral annular calcification (MAC), which is usually observed in advanced age, is associated with increased risk of AF and cardio-embolic stroke in the general population. Objectives: This study was performed to assess whether presence of MAC and its severity predict cardio-embolic stroke in elderly patients with first diagnosed AF. Methods: In this cross-sectional study, 72 elderly patients suffering from acute cardio-embolic stroke with first diagnosed AF and 79 elderly control group patients with first diagnosed AF and without stroke were investigated. A parasternal short-axis view at the level of the mitral annulus was used for MAC measurements. The severity of MAC was measured from the anterior to posterior edge at its greatest width. Results: MAC thicknesses were significantly higher in the stroke group. ROC curve analysis showed that a cut point of 2.5 mm for the value of MAC thickness exhibited 68.1% sensitivity and 77.2% specificity for detecting cardio-embolic stroke in elderly patients with AF. In multivariate logistic regression analysis, MAC thickness (OR = 1.173, 95% CI 1.083-1.270; p < 0.001) was found to be independent predictor of cardio-embolic stroke in elderly patients with AF. Conclusion: MAC thickness may provide useful information for the relevant risk evaluation of elderly patients with AF. Pre-stroke MAC presence and its severity appear to have better clinical value for predicting cardio-embolic stroke in elderly patients with AF, independent from traditional risk factors for stroke.