Islets undergo a number of up-regulatory changes to meet the increased demand for insulin during pregnancy, including increased insulin secretion and β-cell proliferation. It has been shown that elevated lactogenic hormone is directly responsible for these changes, which occur in a phasic pattern, peaking on day 15 of pregnancy and returning to control levels by day 20 (term). As placental lactogen levels remain elevated through late gestation, it was of interest to determine whether glucocorticoids (which increase during late gestation) could counteract the effects of lactogens on insulin secretion, β-cell proliferation, and apoptosis. We found that insulin secretion measured over 24 h in culture and acute secretion measured over 1 h in response to high glucose were increased at least 2-fold by PRL treatment after 6 days in culture. Dexamethasone (DEX) treatment had a significant inhibitory effect on secretion in a dose-dependent manner at concentrations greater than 1 nM. At 100 nM, a concentration equivalent to the plasma corticosteroid level during late pregnancy, DEX inhibited secretion to below control levels. The addition of DEX (> 1 nM) inhibited secretion from PRL-treated islets to levels similar to those produced by DEX treatment alone. Bromodeoxyuridine (10 μM) staining for the final 24 h of a 6-day culture showed that PRL treatment increased cell proliferation 6-fold over the control level. DEX treatment alone (1-1000 nM) did not reduce cell division below the control level, but significantly inhibited the rate of division in PRL-treated islets. YoYo-1, an ultrasensitive fluorescent nucleic acid stain, was added (1 μM; 8 h) to the medium after 1-3 days of culture to examine cell death. Islets examined under confocal microscopy showed that DEX treatment (100 nM) increased the number of cells with apoptotic nuclear morphologies. This was quantified by counting the number of YoYo-labeled nuclei per islet under conventional epifluorescence microscopy. The numbers of YoYo-1-positive nuclei per islet in control and PRL-treated islets were not different after 3 days of culture. However, DEX treatment increased YoYo-1 labeling 7-fold over that in controls. DEX also increased YoYo-1 labeling in PRL-treated islets 3-fold over the control level. These data show that the increased plasma glucocorticoid levels found during the late stages of pregnancy could effectively reverse PRL-induced up-regulation of islet function by inhibiting insulin secretion and cell proliferation while increasing apoptosis.