Islet cells undergo major changes in structure and function to meet the demand for increased insulin secretion during pregnancy, however the nature of the hormonal interactions and signaling events are incompletely understood. Here we used the glucose responsive MIN6 -cell line treated with prolactin (PRL), progesterone (PRG) and dexamethasone (DEX, a synthetic glucocorticoid), which are all elevated during late pregnancy, to study their effects on mechanisms of insulin secretion. DEX alone or in combination with PRL and PRG, inhibited insulin secretion in response to 16 mM glucose-stimulating concentrations. However, in the basal state (3 mM glucose), the insulin levels in response to DEX treatment was unchanged, and three hormones together maintained higher insulin release. There were no changes of protein levels of GLUT2 or glucokinase, however PRL or PRG treatment increased glucokinase activity, while DEX had an inhibitory effect on glucokinase activity. -Ketoisocaproate (-KIC) stimulated insulin secretion was also reduced by DEX alone or in combination with PRL and PRG, suggesting that DEX may inhibit distal steps in the insulin exocytotic process. PRL treatment increased the concentration of intracellular cAMP in response to 16 mM glucose, suggesting a role for cAMP in potentiation of insulin secretion, while DEX alone or in combination with PRL and PRG reduced cAMP levels by increasing phosphodiestase activity. These data provide evidence that PRL and to a lesser extent PRG, which increase in early pregnancy, enhance basal and glucose-stimulated insulin secretion in part by increasing glucokinase activity and by amplifying cAMP levels. Glucocorticoid, which increases throughout gestation, counteracts only glucose-stimulated insulin secretion under high glucose concentrations by dominantly inhibiting glucokinase activity and increasing PDE activity to reduce cAMP levels. These adaptations in the cell may play an important role in maintaining the basal hyperinsulinemia of pregnancy, while limiting the capacity of prolactin and progesterone to promote glucose-stimulated insulin secretion during late gestation.