Background: digoxin use was shown to be associated with an increased risk of cardiovascular events In atrial fibrillation (AF) . We hypothesized that digoxin may affect cardiovascular risk by increasing platelet activation. Methods: This is a post-hoc analysis from an observational prospective study of AF patients treated with vitamin K antagonists. Patients were divided into two groups balanced for age, sex and cardiovascular risk factors: digoxin users (n=132) and non-users (n=388). Urinary excretion of 11-dehydro-thromboxane B 2 (TxB 2 ), a marker of platelet activation and serum digoxin concentration (SDC) were measured. In vitro experiments were performed on platelets from healthy subjects treated with scalar doses of digoxin (0.6 - 2.4 ng/ml). Results: Mean age of digoxin users was 75.2±7.2 years and 47.7% were women. Median overall 11-dehydro-TxB 2 was 105.0 (IQR 60.0-190.0) ng/mg creatinine, and median SDC was 0.65 (IQR 0.40-1.00) ng/ml. Urinary 11-dehydro-TxB 2 and SDC were significantly correlated (rS=0.350, p<0.001). Patients in the upper tertile of SDC showed significantly higher urinary11-dehydro-TxB 2 compared to non-digoxin users (p=0.019). In vitro study showed that digoxin at concentration ≥1.2 ng/ml induced calcium mobilization, arachidonic acid (AA) release, TxB 2 biosynthesis and platelet aggregation, that were inhibited by the calcium chelator Ethylenediaminetetraacetic acid (EDTA) or by the specific phospholipase A 2 (PLA 2 ) inhibitor Arachidonyl trifluoromethyl ketone (ACOOCF3). Conclusion: We found a significant in vivo correlation between SDC and platelet activation. In vitro study showed that supra-therapeutic digoxin concentrations increased platelet aggregation via calcium-related PLA 2 phosphorylation. This finding provides new insights into the association between digoxin use and cardiovascular complications in AF patients.