The aim was to investigate the association between homocysteine (Hcy) and acute coronary syndrome (ACS) and to test the potential moderating role of Mediterranean diet. An age and gender matched case-control study was conducted among 1491 patients with a first ACS event and 3037 adults free of cardiovascular disease (CVD). Adherence to the Mediterranean diet was measured using the MedDietScore (range 0-55). An increase in Hcy levels was associated with a 1% and 3% higher likelihood of ACS among younger (<45 yrs) and middle-aged (45-60yrs) adults (p's < 0.05), but not in older adults (p = 0.13). Moreover, Hcy was associated with 3% (95%CI: 1.01-1.06) increase in the likelihood of ACS among those who did not adhere to the Mediterranean diet. Hence, Hcy is apparently independently associated with ACS among younger and middle-aged individuals. The inverse association between Mediterranean diet adherence and Hcy highlights a disease-preventing effect of the Mediterranean diet on CVD.