BACKGROUND: Circulating linoleic acid (LA) levels have been reported to be associated with various metabolic outcomes. However, the role of LA and its interplay with gut microbiota in gestational diabetes mellitus (GDM) remains unclear. This study aimed to investigate the longitudinal association between circulating LA levels during pregnancy and the risk of GDM, and the potential role of gut microbiota. METHODS: A nested case-control study was conducted within the ongoing Tongji-Huaxi-Shuangliu Birth Cohort in Chengdu, China. Blood and fecal samples were collected during early and middle pregnancy from 807 participants. GDM was diagnosed in middle pregnancy using the International Association of Diabetes and Pregnancy Study Groups criteria. Plasma LA levels were measured using gas chromatography-mass spectrometry, and gut microbiota was analyzed through 16S rRNA gene sequencing and shotgun metagenomic sequencing. A two-sample Mendelian randomization study was conducted using data from the IEU OpenGWAS database and the FinnGen consortium. RESULTS: Elevated plasma LA levels were associated with a lower risk of GDM in both early (P for trend = 0.002) and middle pregnancy (P for trend = 0.02). Consistently, Mendelian randomization analysis revealed that each unit increase in LA was associated with a 16% reduction in GDM risk (odds ratio: 0.84, 95% confidence interval: 0.72, 0.95). In early pregnancy, higher plasma LA levels were correlated with higher adiponectin levels (P < 0.001) and lower levels of triglycerides (P < 0.001), HbA1c (P = 0.04), and C-peptide (P = 0.04). The LA-accociated microbiota mediated the relationship between LA and C-peptide (P = 0.01). Additionally, the inverse association between LA and GDM was modified by Bilophila (P for interaction = 0.03), with a stronger association observed in participants with lower Bilophila levels in early pregnancy. Metagenomic analyses further showed that the LA-associated pathway (D-galacturonate degradation I) and its key enzyme (EC 4.2.1.7) were associated with metabolic traits. CONCLUSIONS: Our study provides evidence for an inverse causal association between plasma LA levels during pregnancy and GDM risk, which is both mediated and modified by gut microbiota.