Ghrelin stimulates growth hormone release and cell proliferation, which strongly supports a significant role for this peptide in the control of growth hormone-releasing adenomas function and growth. Nitric oxide can influence the stimulatory effects of ghrelin on growth hormone secretion in growth hormone-releasing adenomas. However, the effect of nitric oxide (NO) on ghrelin-induced cell proliferation and the mechanism of this effect in the adenoma were not clarified. In this study, we observed that ghrelin, at a concentration of 10⁻⁹ to 10⁻⁶ M, significantly increased BrdU incorporation into rat GH3 cells. A NO donor, S-nitroso-N-acetylpenicillamine (SNAP), blunted basal, and ghrelin-induced cell proliferation. A blocker of NO synthase, Nw-nitro-L-arginine methyl ester hydrochloride (NAME), had no influence on these actions. The activation of extracellular signal-regulated kinase (ERK) 1/2 was examined by western blotting. The results showed that SNAP reduced ghrelin-stimulated ERK1/2 activation but NAME had no influence on this activation. Together, this study indicates that NO inhibited ghrelin-induced cell proliferation by blocking ERK1/2 activation in GH3 cells.