Background <p>Both serum ferritin (SF) and thyroxine (fT4) are associated with the risk of gestational diabetes mellitus (GDM). However, whether and how they interact to modulate GDM risk has yet to be fully explored.</p> Objective <p>This study aimed to determine the effect modification of fT4 on the association of SF and GDM, and to further identify the SF safety range while considering fT4 levels.</p> Methods <p>A retrospective cohort study was conducted in 15,627 pregnant women enrolled from the Southwest region of China between 2018 and 2022. A modified Poisson regression and restricted cubic splines were used to assess the linear and non-linear associations of SF and fT4 with GDM risk in a trimester-specific manner, respectively. Stratified and interaction analyses were performed to evaluate the modification effect of fT4 on SF-GDM relationship. Various sensitivity analyses were conducted.</p> Results <p>Both fT4 and SF were linearly associated with GDM risk during early and mid-pregnancy (all <i>P</i>_association&lt;0.05). Furthermore, fT4 significantly interacted with SF to modulate GDM risk in the first trimester (Pinteraction = 0.012). During early pregnancy, SF was positively associated with GDM risk only at low fT4 levels, with an identified upper risk cut-off for SF of 150 ng/mL (<i>P</i>_trend=0.005). Whereas in mid-pregnancy, high SF (&gt; 15 ng/mL) and low fT4 were independent risk factors for GDM (<i>P</i>_association&lt;0.05).</p> Conclusions <p>This study provides the first evidence that SF interacts with fT4 to modulate GDM risk in a trimester-dependent manner, highlighting the importance of monitoring ferritin levels in the context of thyroid function across different gestational stages for effective GDM prevention.</p>