Obestatin is a 23-amino-acid amidated peptide that is encoded by the ghrelin gene. Previous studies have shown obestatin can modulate the hypothalamic neuronal circuitry that regulates pituitary function, perhaps by modulating the actions of ghrelin. However, the direct actions of obestatin on pituitary function remain controversial. Here, primary pituitary cell cultures from a nonhuman primate (baboon) and mice were used to test the effects of obestatin on pituitary hormone expression and secretion. In pituitary cultures from both species, obestatin had no effect on prolactin, LH, FSH, or TSH expression/release. Conversely, obestatin stimulated proopiomelanocortin expression and ACTH release and inhibited GH expression/release in vitro, actions that were also observed in vivo in mice treated with obestatin. In vitro, obestatin inhibited the stimulatory actions of ghrelin on GH but not ACTH release. The inhibitory effect of obestatin on somatotrope function was associated with an overall reduction in pituitary transcription factor-1 and GHRH receptor mRNA levels in vitro and in vivo as well as a reduction in hypothalamic GHRH and ghrelin expression in vivo. The stimulatory effect of obestatin on ACTH was associated with an increase in pituitary CRF receptors. Obestatin also reduced the expression of pituitary somatostatin receptors (sst1/sst2), which could serve to modify its impact on hormone secretion. The in vitro actions of obestatin on both GH and ACTH release required the adenylyl cyclase and MAPK routes. Taken together, our results provide evidence that obestatin can act directly at the pituitary to control somatotrope and corticotrope function, and these effects are conserved across species.