Tobacco smoking is one of the main causes of cardiovascular disease (CVD). In an observational study in adult Japanese smokers and non-smokers, potential CVD-related biomarkers of effect (BoEff) were measured. The association of cigarette smoking status with the levels of these biomarkers was evaluated. The study objective was to quantify differences in selected, potential CVD-related BoEff, including HDL-cholesterol, high sensitivity C-reactive protein (hsCRP), fibrinogen, and 11-dehydro-thromboxane B2 (11-dehydro-TBX2), between smokers and non-smokers. Adult smokers and non-smokers (ratio of 2:1) of both genders aged 30 years and above, in good general health, were included in this study. The study was conducted at 9 study sites in Japan and was performed according to the principles of good clinical practice. A total of 1098 subjects (731 smokers and 367 non-smokers) were enrolled. Plasma samples from two visits were assessed and data from 1077 of the subjects (716 smokers and 361 non-smokers) were analyzed. A tendency was observed in smokers towards higher levels of 11-dehydro-TXB2 and fibrinogen and lower levels of HDL-cholesterol (based on the review of 95% confidence intervals). Mean blood cell counts of leucocytes, lymphocytes, and monocytes were higher in smokers than in non-smokers. No differences were observed between smokers and non-smokers for hsCRP. In conclusion, the smokers in this study had different levels of some potential CVD-related BoEff compared to non-smokers. Since these may be associated with CVD pathogenesis, these results provide a baseline for further investigation into which BoEff may be predictive of CVD risk related to cigarette smoking.