Associations have been reported between periodontal disease and increased cardiovascular disease risk, as well as between healthy self-reported tooth brushing behavior and reduced cardiovascular disease risk. We examined the association between self-reported tooth brushing behavior and the risk of metabolic syndrome (MetS) using a large medical check-up database. A total of 12,548 medical checkup records from a medical screening center in a city in southwest Japan were assessed. Subjects were aged 30 to 59 years. As lifestyle is associated with both tooth brushing and MetS, many lifestyle variables were considered as potential confounders. Logistic regression model were employed with a list of 127 lifestyle variables. Twenty variables associated with both tooth brushing and MetS were selected for both males and females. Furthermore, final confounding variables were selected by principal component analysis to avoid collinearity problems. The association of tooth brushing and MetS was evaluated for males and females separately by calculating adjusted odds ratios (ORs) with selected confounders plus age. The association between daily tooth brushing frequency and MetS risk for both genders was significant after adjusting for confounders, with the risk of MetS decreasing with increased frequency of daily tooth brushing (adjusted OR = 0.57 (95%Cl:0.48-0.81), 0.50 (95%Cl:0.35-0.71), 0.42 (95%Cl: 0.29-0.61) for males and adjusted OR = 0.65 (95%Cl:0.48-0.87), 0.44 (95%Cl:0.32-0.62) for females). Therefore, oral hygiene, including the frequency of tooth brushing as a significant component, may be an important factor in preventing MetS.