Although aspirin has become an established medicine for cardiac and cerebrovascular diseases, the optimal dose remains unknown. We evaluated the optimal dose of aspirin on platelet activity and endothelial function by administering 11 healthy male volunteers (32 ± 6 years of age) doses of aspirin that were increased in a stepwise manner (0, 81, 162, 330 and 660 mg/day) every 3 days. Platelet activity was assessed as surface P-selectin expression (%) measured by flow cytometry and the platelet aggregation ratio. Endothelial function in the brachial artery was assessed by measuring flow-mediated dilation (FMD) before and after reactive hyperemia. Platelet aggregation and P-selectin expression were significantly and dose-dependently suppressed (81–660 mg), and the FMD ratio tended to increase from 0 to 162 mg, but decreased significantly at 660 mg. In conclusion, although aspirin suppressed platelet activity and even surface P-selectin expression, higher doses worsened endothelial-mediated arterial dilation.