This study aimed to investigate the associations between TMAO and related metabolites in the early pregnancy and risk of GDM.A prospective cohort of 22,302 pregnant women from 2010 to 2012 in Tianjin, China, was used to perform a nested case-control study. 243 women with GDM and 243 women without GDM matched with maternal age (+/-1 year) were used as cases and controls respectively. Conditional logistic regression and restricted cubic spline were used to examine the full-range risk associations between individual TMAOs at the first antenatal care visit with GDM. Trimethylamine ratio (TMAR) was defined as TMA/its precursors while TMAO ratio (TMAOR) was defined as TMAO/TMA. An additive interaction between high TMAR and low TMAOR indicates a state of TMA accumulation while a mathematical interaction between high TMAR and high TMAOR indicates accumulation of TMAO.TMA was linearly associated with GDM while TMA precursors and TMAO were inversely associated with GDM with clear threshold effects, i.e., 16 nmol/mL for TMAO, 200 nmol/mL for betaine, 112 nmol/mL for L-carnitine, and 110 and 270 nmol/mL for cholinechloride (a U-shaped relationship). Co-presence of TMAR >0.35 and TMAOR ≤0.15 was associated with markedly higher odds ratio of 11.16 (5.45-22.8), compared to TMAR >0.35 only (1.71, 0.42-6.95) or TMAOR ≤0.15 only (2.06, 1.09-3.90), with a significant additive interaction. However, the mathematical interaction was non-significant.TMAO metabolites in the early pregnancy were associated with the risk of GDM, while TMA was more likely to play a causal role in GDM.