Background: Vitamin D modulates innate and adaptive immunity, but population-level evidence linking vitamin D status to hepatitis B surface antibody (anti-HBs) positivity is limited. Methods: We conducted a cross-sectional analysis of 4,852 U.S. adults aged 20 years and older from the National Health and Nutrition Examination Survey (NHANES) August 2021–August 2023 cycle with measured serum 25-hydroxyvitamin D [25(OH)D] and anti-HBs. Survey-weighted logistic regression estimated odds ratios (ORs) for anti-HBs positivity by 25(OH)D status (<50 vs ≥50 nmol/L), adjusting for age, sex, race/ethnicity, education, and family income-to-poverty ratio. Effect modification and six sensitivity analyses were examined.Results: The weighted prevalence of anti-HBs positivity was 27.1% (95% CI 25.0–29.3), and 20.0% of adults had 25(OH)D <50 nmol/L. The crude association was null (OR 0.92, 95% CI 0.78–1.08), but after adjustment, 25(OH)D <50 nmol/L was associated with lower odds of anti-HBs positivity (OR 0.72, 95% CI 0.61–0.86). Each 10 nmol/L increase in 25(OH)D was associated with higher odds (OR 1.03, 95% CI 1.00–1.05), and odds were lowest below 30 nmol/L (OR 0.67, 95% CI 0.51–0.87). There was no evidence of nonlinearity. Interaction tests suggested stronger associations in women and younger adults (p = 0.015 and p = 0.022), and sensitivity analyses yielded consistent estimates (ORs 0.67–0.78).Conclusions: Low vitamin D status was independently associated with lower anti-HBs positivity among U.S. adults. The cross-sectional design and the inability to distinguish vaccine-induced from infection-acquired antibodies preclude causal inference.