Thyroid dysfunction is associated with both vitamin D deficiency and iodine; however, it is unclear whether they interact. This study aimed to investigate whether and to what extent the interactions between vitamin D and iodine contribute to the risk of thyroid disorder. Participants (n = 4280) were chosen using multistage, stratified random sampling from Shanghai. Fasting blood was drawn for the 25(OH)D and thyroid parameter tests. Spot urine samples were gathered to test for urine iodine. To evaluate the interactive effects of vitamin D and iodine, crossover analysis was carried out. Pregnant women with a high urinary iodine concentration (UIC) and severe vitamin D deficiency had a significantly higher risk of thyrotropin receptor antibody (TrAb) positivity (odds ratio = 2.62, 95% confidence interval (CI): 1.32, 5.22) in the first trimester. Severe vitamin D deficiency and high UIC interacted positively for the risk of TrAb positivity (relative excess risk due to interaction = 1.910, 95%CI: 0.054, 3.766; attributable proportion = 0.700, 95%CI: 0.367, 1.03). Severe vitamin D deficiency combined with excess iodine could increase the risk of TrAb positivity in pregnant women in the first trimester.