||The activation of STAT3 through functional coupling of Gi-coupled receptors with Gα16 has been demonstrated previously. On the other hand, melatonin is a pleotropic indolehormone, modulating various biological responses mainly through its Gi-coupled melatonin membrane receptors. The aim of this research is to examine the STAT3 activation through functional coupling of melatonin receptors with Gα16, to define the signaling intermediates involved, and any biological significance of this melatonin-induced STAT3 response. Immunoblotting with inhibitor treatment showed that the melatonin analogue 2-iodomelatonin can activate STAT3 through its stimulation of the melatonin receptors and Gα16 stably expressed in HEK293 cells in a PTX-insensitive manner. In addition, multiple signaling intermediates were involved in such response, including protein kinase C, protein kinase D (PKD), Src-family tyrosine kinases, JAK2, p38, MEK, as well as the production of autocrine factors. Apart from this, inhibitior treatment with PKC-specific inhibitor RO320432 at 1 μM and Gö6976 at 100 nM revelaed that PKCε may be the upstream kinase of PKD, and possible link between PKD and STAT3 was also demonstrated.