Abstract Introduction During pregnancy, maternal cortisol levels are increased 3-fold by the third trimester. The enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD, isoforms 1 and 2) regulates the balance between cortisol and cortisone levels. Perfluoroalkyl substances (PFAS) have been reported to inhibit 11β-HSD1 and more potently 11β-HSD2, which could lead to reduced levels of cortisol and more extensively cortisone. Aim The aim of this work is to investigate a possible effect of early pregnancy PFAS exposure on late pregnancy activity of 11β-HSD1 and 11β-HSD2 assessed by cortisol and cortisone levels in diurnal urine (dU) and blood samples. Methods This study is part of the prospective cohort study, Odense Child Cohort (OCC). A total of 1628 pregnant women had serum (S) concentrations of 5 PFAS (perfluorooctanoic acid [PFOA], perfluorooctane sulfonic acid [PFOS], perfluorohexane sulfonic acid [PFHxS], perfluorononanoic acid [PFNA], and perfluorodecanoic acid (PFDA)) measured in the first trimester (median gestational week, GW 11). dU cortisol and cortisone (n = 344) and S-cortisol (n = 1048) were measured in the third trimester (median GW 27). Results In multiple regression analyses, a 2-fold increase in S-PFOS was significantly associated with lower dU-cortisone (β = –9.1%, P < .05) and higher dU-cortisol/dU-cortisone (dU-C/C) (β = 9.3%, P < .05). In crude models, a doubling in PFOS, PFOA, PFHxS, and PFNA concentrations were associated with a significant increase in S-cortisol; however, these associations became insignificant after adjustment. Conclusion Early pregnancy maternal S-PFAS were inversely associated with late pregnancy dU-cortisone, indicating reduced activity of 11β-HSD2.