Gestational diabetes mellitus (GDM) is a growing concern for public health, affecting approximately 20% of pregnancies globally. This underscores an urgent need for improved diagnostic and management strategies. This study examines the relationship between trimethylamine N-oxide (TMAO) and its precursor trimethylamine (TMA) levels and GDM, aiming to deepen our understanding of GDM's pathophysiology and identify novel therapeutic targets.The meta-analysis adhered to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines. The PubMed, Scopus, Web of Science, and the Cochrane Library electronic databases were comprehensively searched up to 11 July 2024.The analysis included five studies, encompassing a total of 1,726 participants. The studies reported TMAO levels among GDM and non-GDM patients. The reported TMAO levels among GDM and non-GDM patients were 57.66 ± 42.2 and 47.94 ± 30.86, respectively (SMD = -0.49; 95%CI: -2.69 to 1.71; p = 0.66). However, TMA levels in the GDM group (224.28 ± 39.88) were statistically higher than in the non-GDM group (124.05 ± 21.93; SMD = 3.11; 95%CI: 2.84 to 3.37; p<0.001).The best available evidence indicates that while TMA levels are significantly increased in GDM, TMAO does not seem to have a diagnostic role in gestational diabetes mellitus. More prospective trials evaluating TMA and TMAO values among pregnant women with gestational diabetes mellitus are required.