In a recent clinical study, we investigated the long-term effects of smoking and moist snuff consumption using a panel of biomarkers of effect (BioEff) indicative of inflammation, oxidative stress and lipid metabolism. In this, cross-sectional study, we enrolled generally healthy adult males into three cohorts: non-tobacco consumers (NTC) and long-term smokers and moist snuff consumers (MSC). Twenty-four hour urine samples and matching plasma samples were collected from subjects who abstained overnight from both food and tobacco. Compared to NTC and MSC, smokers exhibited elevated levels of biomarkers associated with oxidative stress (urinary isoprostanes and leukotriene E4), inflammation (white blood cell count), and platelet activation (thromboxane metabolites). A trend for elevated levels of several lipids and lipoprotein markers were observed in smokers. Statistically significant higher levels were only observed for apolipoprotein B100 and oxidized low-density lipoprotein in smokers relative to NTC and MSC. Thus, alterations in BioEff suggesting inflammation, oxidative stress and altered lipid metabolism were detected in smokers compared to the non-smoking cohorts. These findings are generally consistent with a previously conducted RJRT study which showed similar BioEff changes in oxidative stress and inflammatory pathways in smokers relative to MSC, with exceptions in some biomarkers. Collectively, our data suggest smokers, relative to the non-smoking cohorts, exhibit perturbations in pathways that could contribute to the development of smoking-related diseases. In summary, our findings are in agreement with existing epidemiological data which show the reduced harm from smokeless tobacco consumption compared to smoking, with no-tobacco-use being the least risky. The BioEff evaluated in this study are likely to be useful in future assessments of the health effects of new tobacco products.