Human umbilical vein endothelial cells (HUVECs) undergo apoptosis in response to serum deprivation. We show that the nonspecific mineralocorticoid receptor (MR) antagonist, spironolactone, protects from caspase-3 activation induced by serum deprivation in contrast to the selective MR antagonist, eplerenone, that is non-protective. We also demonstrate that progesterone, hydrocortisone and dexamethasone all protect HUVECs from serum-deprivation induced caspase-3 activation, whereas aldosterone and dihydrotestosterone have no effect. Spironolactone has been demonstrated to display agonist activity only to the progesterone receptor and we additionally show that spironolactone and progesterone, but not eplerenone, inhibit mitochondrial cytochrome c release and cleavage of nuclear poly (ADP-ribose) polymerase (PARP) and increase cell viability. Additionally, the PR antagonist mifepristone (RU486) partially blocked the inhibitory effect of both spironolactone and progesterone on caspase-3 activation, cytochrome c release and nuclear PARP cleavage. Nitric oxide protects HUVECs from apoptosis in response to various stimuli including serum-deprivation; however, the nitric oxide synthase inhibitor N-monomethyl-L-arginine, did not abolish inhibition of caspase-3 activation or PARP cleavage by spironolactone. Thus, we demonstrate that spironolactone protects HUVECs from serum-deprivation induced apoptosis by inhibition of caspase-3 activity, cytochrome c release and PARP cleavage by a NO-independent mechanism; further, this effect is likely mediated by the agonist properties of spironolactone toward the progesterone receptor.