Adrenal medulla chromaffin cells are neuroendocrine and modified sympathetic ganglion cells. Catecholamines released from chromaffin cells mediate the "fight-or-flight" response, or alert reaction against dangerous conditions. Here we report that short-term treatment with glucocorticoids, released from adrenal cortex cells in response to chronic stress, inhibits activity-dependent potentiation (ADP) of catecholamine release. First, short-term treatment with dexamethasone (DEX), a synthetic glucocorticoid, reduces ADP in a concentration-dependent manner (IC₅₀: 324.2 ± 54.5 nM). The inhibitory effect of DEX is not reversed by RU-486 treatment, suggesting that the rapid inhibitory effect of DEX on ADP of catecholamine release is independent of glucocorticoid receptors. Second, DEX treatment reduces the frequency of fusion between vesicles and plasma membrane without affecting calcium influx. DEX disrupts activity-induced vesicle translocation and F-actin disassembly, thereby leading to inhibition of the vesicle fusion frequency. Third, we provide evidence that DEX reduces F-actin disassembly via inhibiting phosphorylation and translocation of MARCKS (myristoylated alanine-rich C kinase substrate) and its upstream kinase PKC-epsilon. Altogether, we suggest that glucocorticoids inhibit ADP of catecholamine release by decreasing MARCKS phosphorylation, which inhibits F-actin disassembly and vesicle translocation.