Background: Vitamin D is hypothesized to reduce risk for tooth loss via its influence on bone health, inflammation, and the immune response. The association between plasma 25‐hydroxyvitamin D [25(OH)D] concentrations and prevalence and 5‐year incidence of tooth loss in a cohort of postmenopausal females was examined.Methods: Participants underwent oral examinations at study baseline (1997 to 2000) and follow‐up (2002 to 2005) to determine the number of missing teeth and 5‐year incidence of tooth loss, respectively. At both visits, females self‐reported reasons for each missing tooth. At baseline, 152 females reported no history of tooth loss, and 628 were categorized as reporting a history of tooth loss as a result of periodontal disease (n = 70) or caries (n = 558) (total n = 780). At follow‐up, 96, 376, 48, and 328 females were categorized into the aforementioned categories related to tooth loss (total n = 472). Logistic regression was used to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) for tooth loss by category of baseline 25(OH)D (nmol/L) concentrations. Models were adjusted for age, income, smoking status, frequency of dental visits, waist circumference, and recreational physical activity. P value for trend was estimated using continuous concentrations of 25(OH)D.Results: Among females with 25(OH)D ≥50 (adequate vitamin D status) compared to <50 nmol/L (deficient/inadequate), the adjusted ORs were 1.24 (95% CI = 0.82 to 1.87), P‐trend = <0.05 for the history (prevalence) of tooth loss resulting from periodontal disease or caries and 1.07 (95% CI = 0.62 to 1.85), P‐trend = 0.11 for the incidence of tooth loss resulting from periodontal disease or caries. No statistically significant association was observed between 25(OH)D and the history or incidence of tooth loss caused by periodontal disease. An increased odds of the history of tooth loss attributable to caries was observed with increasing concentrations of 25(OH)D (P‐trend = <0.05) but was not confirmed in prospective analyses.Conclusion: In this cohort of postmenopausal females, the data do not support an association between vitamin D status and tooth loss.