We performed a cross-sectional, multicentre study in Japan to detect the differences in biomarkers of exposure and cardiovascular biomarkers between smokers and non-smokers. Several clinically relevant cardiovascular biomarkers differed significantly between smokers and non-smokers, including lipid metabolism (high-density lipoprotein cholesterol concentrations - lower in smokers), inflammation (fibrinogen and white blood cell count - both higher in smokers), oxidative stress (8-epi-prostaglandin F2α - higher in smokers) and platelet activation (11-dehydro-thromboxane B2 - higher in smokers) (p ≤ 0.0001). These results provide further evidence showing that cardiovascular biomarkers can discriminate smokers from non-smokers, and could be used to evaluate the risks associated with tobacco products.