OBJECTIVE A better identification of the role of vitamin D in the thrombotic process of acute coronary syndrome (ACS) will help increase the therapeutic options for this important clinical condition. There is little published evidence that 25-hydroxy vitamin D (25(OH)D) serum levels can associate with platelet function and risk of thrombosis. METHODS This prospective study was conducted on 200 patients with a diagnosis of ACS, including patients with unstable angina (UA), non-ST segment elevation myocardial infarction (NSTEMI), and ST segment elevation myocardial infarction (STEMI). In addition to demographics and angiographic data, serum concentrations of 25(OH)D and MPV were recorded in all patients. RESULTS The types of ACS were STEMI (35%), NSTEMI (25%), and UA (40%). The concentrations of 25(OH)D in patients with UA, NSTEMI, and STEMI were 23.53 ± 13.26, 19.25 ± 8.09 and 14.60 ± 8.24 ng/mL respectively (P < 0.001), and the values of MPV were 9.83 ± 1.35, 10.30 ± 1.21, and 11.56 ± 1.38 fL, respectively (P < 0.001). There was a negative correlation between 25(OH)D and MPV (R = -0.320, P < 0.0001). ROC analysis illustrated a moderate predictive value (AUC 0.706; 95% CI, 0.63-0.72) in identifying the discrimination threshold of MPV (≥ 9.90 fL) for vitamin D deficiency (<20 ng/mL). CONCLUSIONS The current study shows an inverse association between MPV and vitamin D levels in ACS patients, especially in the subgroup of STEMI. These findings propose the effect of vitamin D on platelet size and function, suggesting its role in thrombosis and hemostasis, which might explain the link between vitamin D deficiency and cardiovascular diseases.