Objective: Low circulating prolactin is a potential marker of metabolic risk during pregnancy. We aimed to investigate associations between prolactin and glucose status in pregnant women with and without gestational diabetes mellitus (GDM) or polycystic ovary syndrome (PCOS). Design: Prospective observational cohort study. From the Odense Child Cohort, 1497 pregnant women were included. Methods: Blood samples were assessed during first, second (prolactin, hemoglobin A1c (HbA1c)) and third trimester (fasting prolactin, testosterone, HbA1c, insulin, glucose). Oral glucose tolerance test (OGTT) was performed around gestation week 28 in 350 women with risk factors for GDM and in 272 randomly included women. GDM was defined by 2-h plasma glucose ≥9.0 mmol/L. Results: The median (IQR) prolactin increased from 633 (451-829) mIU/L in first-second trimester to 5223 (4151-6127) mIU/L at third trimester. Prolactin was inversely associated with HbA1c in first (r = -0.19, P < 0.001) and third trimester (r = -0.07, P = 0.014). In third trimester, women with GDM (n = 37; 6.0%) had lower prolactin compared to women without GDM (4269 vs 5072 mIU/L, P = 0.004). Third trimester prolactin multiple of the median (MoM) was inversely associated with risk of GDM in multivariate regression analysis (OR 0.30, P = 0.034). PCOS was diagnosed in 10.0% (n = 146). Early pregnancy prolactin MoM was positively associated to PCOS diagnosis (OR 1.38, P = 0.051). Conclusions: Low prolactin levels during pregnancy were associated with higher HbA1c and risk of GDM. A diagnosis of PCOS was associated with higher early pregnancy prolactin levels.