Previous studies have reported an association between iron deficiency (ID) and increased thyroid peroxidase antibody (TPO-ab) during early pregnancy. The objective of this study was to explore the relationship between ID and thyroid dysfunction, as well as thyroid autoantibodies, during the second trimester of pregnancy. A total of 1592 pregnant women (13-28 weeks gestation) were enrolled in this cross-sectional study. According to serum ferritin (SF) concentrations, they were divided into ID (SF <20 μg/L) or non-ID (SF ≥20 μg/L) groups. Logistic regression analysis was used to evaluate the association between ID and subclinical hypothyroidism (thyroid-stimulating hormone [TSH] >4.0 mIU/L and free thyroxine [FT4] within the reference range) and thyroid autoimmunity. The prevalence of ID was 23.43% (373/1592). Compared with the non-ID group, the ID group had lower FT4 levels (13.94 [8.91-29.82] vs 14.63 [8.22-47.24] pmol/L, p<0.001]) and higher TSH levels (1.85 [0.01-7.84] vs 1.69 [0.01-10.2] mIU/L, p<0.05). Logistic regression analysis confirmed ID as a risk factor for increased thyroglobulin antibody (TG-ab) (odds ratio 1.974; 95% confidence interval 1.065, 3.657; p<0.05), but not for subclinical hypothyroidism or increased TPO-ab. ID is associated with increased TG-ab during the second trimester of pregnancy.