Background: Low serum vitamin D levels have been associated with several autoimmune diseases, but their association with thyroid autoimmunity is unclear. We evaluated the association of serum vitamin D levels with the prevalence of autoimmune thyroid disease (AITD). Methods: Our cross-sectional study included subjects who underwent routine health checkups, which included assays of serum 25-hydroxy vitamin D3 [25(OH)D3] and anti-thyroid peroxidase antibody (TPO-Ab), as well as thyroid ultrasonography (US) between 2008 and 2012 at the Asan Medical Center. We defined AITD according to the levels of TPO-Ab and US findings. Results: A total of 6685 subjects (58% male; 42% female) were enrolled for this study. Overall prevalence of TPO-Ab positivity and both TPO-Ab/US positivity were 10.1% (6.3% male; 15.3% female) and 5.4% (2.3% male; 9.7% female) respectively. In female subjects, mean serum 25(OH)D3 levels were significantly lower in the TPO-Ab(+) (22.0 vs. 23.5 ng/mL, p = 0.030) and TPO-Ab(+)/US(+) groups (21.6 vs. 23.4 ng/mL, p = 0.027) compared with the control group, respectively. According to the levels of serum 25(OH)D3, the prevalence of TPO-Ab positivity (21.2%, 15.5%, and 12.6% in deficient, insufficient, and sufficient group, respectively; p = 0.001) and both TPO-Ab and US positivity (14.7%, 9.9%, and 7.1% in deficient, insufficient, and sufficient group, respectively; p < 0.001) decreased in female subjects. Interestingly, this pattern was significant only in pre-menopausal women ( p = 0.003 and p < 0.001; respectively), but not in postmenopausal women. Multivariate analysis indicated that the adjusted odds ratios (