Objective <p>The objective is to investigate the differences in urinary organic acid (OA) profiles and metabolism between healthy control (HC) pregnant women and those with gestational diabetes mellitus (GDM) during the second trimester and third trimester of pregnancy.</p> Methods <p>A total of 66 HC pregnant women and 32 pregnant women with GDM were assessed for 107 hydrophilic metabolites in urine samples collected during the second and third trimester of pregnancy using tandem mass spectrometry. The urine OA profiles for each group were obtained, and metabolomic analysis and discussion were conducted.</p> Results <p>This study identified a total of 50 metabolic biomarkers. In the third trimester of pregnancy, short-chain dicarboxylic acids (DCAs) and tryptophan (Trp)-related metabolites were significantly upregulated in the urine of both the HC group and the GDM group. Comparatively, the glycine (Gly) levels and related synthetic precursor metabolites were lower in the GDM2 group. The overall dietary polyphenol metabolic intermediates level in the GDM group was lower than in the HC group. Among the pathways enriched for differentially expressed metabolites, the predominant metabolic pathway in the GDM group was the citric acid cycle. In contrast, in the HC group, it was the metabolism of alanine, aspartate, and glutamate.</p> Conclusions <p>The study reveals the differences in metabolomics between pregnant women with HC and those with GDM, identifying several metabolites associated with the occurrence and development of GDM. Demonstrating the presence of abnormal mitochondrial and peroxisomal functions at the metabolite level in GDM will contribute to future exploration of the condition.</p>