Background: Toothbrushing is a health-related lifestyle habit and has been reported to contribute not only to oral health but also to some parameters of general health; however, little research has been conducted to understand the association of the frequency and timing of toothbrushing with the development of comprehensive metabolic abnormalities, with consideration of oral health condition. In this study, using longitudinal data, we examined this association in Japanese adults, adjusting for periodontal condition. Methods: A 5-year longitudinal study was performed with 4,537 participants between 35 and 64 years old who underwent an annual dental examination in both 2003 and 2008. Data about toothbrushing habits and metabolic abnormalities, such as obesity, hyperglycemia, diabetes, hypertension, hypertriglyceridemia, and low levels of high-density lipoprotein-cholesterol, were analyzed using Poisson regression analysis. Results: The percentage of participants with a toothbrushing frequency ≤1 time=day was 29.4%, and that for those not brushing their teeth at night was 21.4%. The incidences of obesity and hyperglycemia after 5 years were 5.5% and 28.4%, respectively. A toothbrushing frequency ≤1 time=day was associated with development of obesity (prevalence rate ratio [PRR] 1.77; 95% confidence interval [CI], 1.12-2.80), after adjusting for periodontal condition and potential risk factors. A significant association between not brushing teeth at night and hyperglycemia (PRR 1.30; 95% CI, 1.02-1.66) was observed in participants with toothbrushing frequency of 1 time=day. No association was found between toothbrushing habits and other metabolic abnormalities. Conclusions: This study suggests that toothbrushing habits are associated with the development of obesity and hyperglycemia.