Previous studies have indicated low serum carotenoid concentrations among cigarette smokers and/or alcohol drinkers, but little is known about the interaction of smoking and drinking with serum carotenoids. We tested the hypothesis that smoking and drinking reduce serum carotenoid concentrations synergistically. A total of 1073 subjects (357 male and 716 female) who had received health examinations in the town of Mikkabi, Shizuoka Prefecture, Japan, participated in the study. The subjects were divided into six groups according to alcohol intake (non-drinkers, < 1 g/d; light drinkers, ≥ 1, < 25 g/d; moderate-to-heavy drinkers, ≥ 25 g/d) and smoking status (non-smokers and current smokers). The dietary intakes and serum concentrations of six carotenoids (lycopene, α-carotene, β-carotene, lutein, β-cryptoxanthin and zeaxanthin) within each group were evaluated cross-sectionally. The dietary intakes of all carotenoids did not differ in the six groups after adjusting for age and sex. The multivariate-adjusted means of the serum carotenoid concentrations in non-drinkers did not differ between non-smokers and current smokers. In contrast, the adjusted means of serum α-carotene, β-carotene and β-cryptoxanthin were significantly lower than those with increased alcohol intake, and these lower serum carotenoids among alcohol drinkers were more evident in current smokers than in non-smokers. Serum lycopene of moderate-to-heavy drinkers was significantly lower than that of non-drinkers, but it was not influenced by smoking. Neither smoking nor drinking was associated with the serum concentrations of lutein and zeaxanthin. These results suggest that smoking and drinking may reduce the serum α-carotene, β-carotene and β-cryptoxanthin concentrations in a synergistic manner.