Smoking showed to effect on platelet aggregation and the response to anti-platelet drugs. The arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) increases platelet aggregation and blood pressure. In this study, we investigated the influence of cigarette smoking on plasma 20-HETE levels and protein expression of 20-HETE producing enzyme CYP4A11 in isolated platelets from smoker (N=6) and non-smoker (N=6) volunteers. The protein expression and 20-HETE levels were analyzed using immunublot and high-performance liquid chromatography with Mass spectrometry assays, respectively. The results showed that plasma 20-HETE level was significantly higher among smokers than non-smokers (t test, pvalue<0.05). The protein expression of CYP4A11 was significantly higher (t test, p-value<0.05) in platelets isolated from smokers, which was correlated with in vitro higher 20-HETE production, than non-smoker platelets. We concluded from this study that cigarette smoking increased the level of platelet activator 20-HETE through increasing the protein expression of CYP4A11. These findings may increase our understanding of the molecular mechanism of smoking in platelet aggregation and in the variation of anti-platelet drug response induced by smoking.