Serum concentrations of progesterone sulphates are raised in intrahepatic cholestasis of pregnancy (ICP), the commonest pregnancy-specific liver disease. Women with ICP have increased rates of gestational diabetes mellitus (GDM). We hypothesised that raised progesterone sulphates may modulate glucose homeostasis. Progesterone sulphates were assayed in serum samples from participants of the hyperglycaemia and adverse pregnancy outcomes (HAPO) study (n=7994), and from women with GDM (n=19) matched with healthy controls (n=39), using ultra-performance liquid chromatography and mass spectrometry. Islets were isolated from female C57BL/6 mice or obtained from human organ donations. Static incubation was performed in both mice and human islets to assess insulin secretion in the presence of progesterone sulphates. As progesterone sulphates can bind the bile acid receptors, FXR and TGR5, islets were studied from Fxr−/− and Tgr5−/−mice. Analysis of insulin was by radioimmunoassay. Serum samples from the HAPO study demonstrated significantly lower progesterone sulphates in women with higher fasting plasma glucose. Reductions were seen in PM3S, PM3DiS (P<0.05), epiallopregnanolone sulphate (PM5S) and allopregnanolone sulphate (PM4S) (P<0.01) and pregnanolone (PM6S) (P<0.001). Similarly, women diagnosed with GDM had reduced serum progesterone sulphate concentrations, in particular PM5S (P<0.05). In islets, 50 μM PM5S was shown to increase glucose stimulated insulin secretion by at least 2-fold in both mouse and human islets at 20 mM glucose concentrations (P<0.001). This effect was not abolished from islets obtained from Fxr−/− or Tgr5−/− mice. In conclusion, progesterone sulphates are reduced in the serum of women with GDM and increase glucose stimulated insulin secretion. This is not mediated by Fxr or Tgr5. The increased rate of GDM seen in ICP women could be linked to progesterone sulphate mediated insulin release.