BACKGROUND: Perfluoroalkyl substances (PFAS) are chemicals with endocrine disrupting properties. Experimental studies indicate that PFAS have estrogenic effects by inducing aromatase activity. Sex hormone-binding globulin (SHBG) is a marker of the balance between estrogen and testosterone, as estrogen stimulates and testosterone inhibits SHBG production. OBJECTIVE: To investigate associations between maternal PFAS concentrations and levels of SHBG and testosterone in pregnancy. METHODS: In Odense Child Cohort (OCC), concentrations of PFAS: perfluorohexane sulfonic acid (PFHxS), perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) were measured in 1611 eligible women at median gestational week (GW) 12 (25th, 75th percentile: 10, 15). Among these, levels of sex hormone-binding globulin (SHBG), calculated free testosterone (Free-T), free androgen index (FAI), and total testosterone (TT) were assessed in 1048 at median GW 29 (25th, 75th percentile: 28, 30). Associations between PFAS concentrations and levels of SHBG and testosterone were estimated using multiple linear regression models. The effect of combined exposure to PFAS was also assessed via Quantile G-Computation. RESULTS: A doubling in PFOS concentration was associated with an increment in SHBG concentration by 2.29% (95%CI: 0.04%, 4.59%) in adjusted analyses. PFOS exposure in the third tertile, as compared to the first tertile, significantly increased SHBG concentrations by 4.60% (95%CI: 0.82%, 8.53%). No significant association was demonstrated between PFAS and TT, however, a non-significant inverse association was found between PFAS and Free-T and FAI. Combined PFAS exposure was non-significantly associated with an increase in SHBG, and decrease in Free-T, FAI, and TT. CONCLUSION: PFOS exposure was associated with an increase in the proportion of estrogen effects to androgen effects, assessed by higher SHBG concentrations, in pregnant women. Estrogenic effects during pregnancy may have implications on offspring neural, metabolic, and endocrine development, hence supporting the necessity of a follow-up of offspring.