Previous studies have suggested that pesticide exposure and gut microbiome alterations are associated with gestational diabetes mellitus (GDM) risk. Understanding the complex interactive effect of these factors on GDM is essential. In a cohort of 852 pregnant women, we assessed pesticide levels in serum and analyzed the gut microbiota using 16S rRNA and shotgun metagenomic sequencing. We explored the interactions between pesticides and gut microbiota, assessed their roles in GDM development, and proposed a predictive model based on identified biomarkers. We identified an environmental risk score (ERS), denoting the pesticide mixture level significantly associated with GDM, with the gut microbiota, particularly involving the