Ghrelin is a stomach-derived orexigenic hormone whose levels in circulation are altered by energy availability. Like ghrelin, the glucotropic hormone glucagon increases in the fasting state and serves to normalize energy levels. We hypothesized that glucagon can directly stimulate stomach ghrelin production. To verify this hypothesis, we used a primary culture of dispersed rat stomach cells. We first demonstrated that stomach ghrelin cells express the glucagon receptor (GluR). Glucagon (1-100 nM) significantly stimulated ghrelin secretion and proghrelin mRNA expression, and co-incubation with a GluR inhibitor prevented glucagon's action. The MAP kinase inhibitor (PD98058) reduced the glucagon-stimulated ghrelin secretion and proghrelin mRNA expression. Furthermore, glucagon treatment increased the phosphorylation of ERK1/2. Glucagon also increased intracellular cAMP levels, and inhibition of adenylate cyclase reduced glucagon's effect on ghrelin secretion. Surprisingly, inhibiting protein kinase A (PKA) (using H89 and phosphorothioate [Rp]-cAMP) did not prevent glucagon-stimulated ghrelin secretion. Instead, inhibiting the exchange protein activated by cAMP (EPAC) with Brefeldin-A was able to significantly reduce glucagon-stimulated ghrelin secretion. Furthermore, the EPAC agonist (8-pCPT) significantly stimulated ghrelin secretion. Depleting endoplasmic reticulum calcium stores or blocking voltage-dependant calcium channels prevented glucagon stimulated ghrelin secretion. Finally, co-incubation with the sympathetic neurotransmitter norepinephrine potentiated the glucagon stimulation of ghrelin secretion. Our findings are the first to show a direct link between glucagon and stomach ghrelin production and secretion and highlight the role of MAPK, the PKA-independent EPAC pathway, and the synergy between norepinephrine and glucagon in ghrelin release.