Perfluoroalkyl substances (PFASs) are persistent chemicals with suspected endocrine disrupting abilities applied in consumer products. PFASs have potentially modulating effects on glucose homeostasis. Insulin resistance prevails during third trimester of pregnancy, and this challenge of glucose homeostasis may reveal putative effects of PFAS concentrations on glycemic status.To investigate associations between five serum PFASs and glucose-related outcomes in pregnant Danish women based on their risk of gestational diabetes mellitus (GDM).In the prospective Odense Child Cohort serum concentrations of five PFASs - perfluorohexane sulfonic acid (PFHxS), perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) - were measured at median gestational week (GW) 11 in pregnant women. An oral glucose tolerance test (OGTT) was performed at GW 28. The statistical analysis was conducted among 158 women with high GDM risk and 160 women with low GDM risk matched by gestational age. Multiple linear regression models were performed to estimate associations between PFAS concentrations and glucose, insulin, C-peptide, homeostatic model of assessment of insulin resistance (HOMA-IR) and beta cell function (HOMA-%β), and insulin sensitivity (Matsuda index) during the 2-h OGTT.In women with high risk for GDM, a two-fold increase in PFHxS concentration was significantly associated with increased fasting glucose, fasting insulin and HOMA-IR after adjusting for age, parity, educational level and pre-pregnancy BMI. Adjusting for the same confounders, a doubling in PFNA concentration was associated with higher fasting insulin and HOMA-%β. In women with low GDM risk, no associations were found between PFAS concentrations and glucose-related outcomes.PFHxS and PFNA concentrations were associated with impaired glycemic status in metabolically vulnerable pregnant women and might further enhance the risk of developing GDM.