Epidemiologic studies of risk and protective factors for poor oral health have mostly been among children and adults, seldom focusing on adolescents. The aims of this study were to examine whether serum cotinine, a marker of tobacco exposure, and serum vitamin D are independently associated with dental caries experience among adolescents.Cross-sectional data from 2579 adolescent participants in the 2005-2008 National Health and Nutritional Examination Survey were analysed. The exposures, cotinine and vitamin D, were measured in serum while dental caries experience was determined by screening examination, and a case defined as having ≥1 decayed or filled teeth. Survey-adjusted logistic regression estimated prevalence odds ratios (POR) and 95% confidence intervals (CI) between the respective exposures and outcome.The overall median (IQR) serum cotinine level was 0.15 ng/mL (0.04, 1.80) while the mean (SE) serum vitamin D concentration was 68.2 nmol/L (1.36). In contrast to participants with serum cotinine ≤3 ng/L, those with levels >3 ng/L had covariate-adjusted estimate of 1.69 (1.16, 2.47) for caries experience. Each additional mean (ng/L) serum cotinine level was associated with an 8% (95% CI = 1.01, 1.16) greater adjusted prevalence odds of having caries experience. In contrast to participants with vitamin D ≥ 50 nmol/L, those with levels of30- < 50 nmol/L and <30 nmol/L had non-statistically significant adjusted estimates of 1.02 (0.72, 1.44) and 1.23 (0.70, 2.16) respectively for caries experience.Serum cotinine is associated with dental caries experience among adolescents and while deficient (<30 nmol/L) levels of vitamin D appear to be associated with greater prevalence odds of caries experience, this association is inconclusive.