Serotonin (5-hydroxytryptamine (5-HT)) is an important neurotransmitter that regulates multiple events in the central nervous system. Many of the 5-HT functions are mediated via G protein-coupled receptors that are coupled to multiple heterotrimeric G proteins, including Gs, Gi, and Gq subfamilies (Martin, G. R., Eglen, R. M., Hamblin, M. W., Hoyer, D., and Yocca, F. (1998) Trends Pharmacol. Sci. 19, 2-4). Here we show for the first time that the 5-hydroxytryptamine 4(a) receptor (5-HT4(a)) is coupled not only to heterotrimeric Gs but also to G13 protein, as assessed both by biochemical and functional assays. Using reconstitution of 5-HT4(a) receptor with different G proteins in Spodoptera frugiperda (Sf.9) cells, we have proved that agonist stimulation of receptor-induced guanosine 5′-(3-O-thio)triphosphate binding to Gα13 protein. We then determined that expression of 5-HT4(a) receptor in mammalian cells induced constitutive- as well as agonist-promoted activation of a transcription factor, serum response element, through the activation of Gα13 and RhoA. Finally, we have determined that expression of 5-HT4(a) receptor in neuroblastoma × glioma NIE-115 cells cause RhoA-dependent neurite retraction and cell rounding under basal conditions and after agonist stimulation. These data suggest that by activating 5-HT4(a) receptor-G13 pathway, serotonin plays a prominent role in regulating neuronal architecture in addition to its classical role in neurotransmission.