This study examined the effect of GHRP-6, a known GHSs receptor agonist, on the phosphorylation of cAMP-responsive element-binding protein (CREB) and the underly mechanism. GH3 cells were cultured and subjected to different treatments as follows: GHRP-6, GHRP-6 plus GHRH, phorbol ester (PMA), an activator of PKC, alone or in combination with GHRP-6, Gö6983, a general inhibitor of PKCs, in the presence or absence of GHRP-6, rottlerin, an inhibitor of PKCs, alone or plus GHRP-6. The cells were transiently transfected with PKCsigma-specific siRNA and then treated with GHRP-6. GH level was measured by enzyme-linked immunosorbent assay (ELISA). The expression of phosphor-CREB, PKCsigma, PKCtheta and phosphor-PKCsigma was determined by Western blotting. The results showed that GHRP-6 stimulated GH secretion in both time- and dose-dependent manners and enhanced the effect of GHRH on GH secretion. GHRP-6 was also found to induce CREB phosphorylation. Moreover, GH secretion was enhanced by the PKC activator PMA and reduced by the PKC inhibitors (Gö6983, rottlerin) and knockdown of PKCsigma. PKCsigma could be activated by GHRP-6. It is concluded that PKC, especially PKCsigma, mediates CREB phosphorylation and GHRP-6-induced GH secretion.