Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter involved in a wide range of central and peripheral physiological functions. A number of different G-protein coupled 5-HT receptors are known to sensitively modify different neuronal networks by their specific action on synaptic transmission and postsynaptic excitability. Here we show for the first time that the 5-HT4 receptor is coupled not only to the heterotrimeric Gs, but also to G13 protein. Activation of this signaling pathway results in RhoA-mediated modulation of gene transcription and in reorganization of the actin cytoskeleton. We also demonstrated that serotonin receptor 5-HT7 can activate heterotrimeric G12 protein in addition to the Gs protein, leading to the selective activation of small GTPases RhoA and Cdc42. Agonist-dependent activation of the 5-HT7 receptor induced pronounced filopodia formation via a Cdc42-mediated pathway paralleled by RhoA-dependent cell rounding. Analysis of mouse hippocampal neurons demonstrated that activation of the endogenous 5-HT7 receptors significantly increased neurite length, whereas stimulation of the endogenous 5-HT4 receptors lead to a pronounced decrease in the length and number of neurites. These data demonstrate distinct roles for the 5HT₇R/G12 and 5-HT₄R/G13 signaling pathways in the neurite outgrowth/retraction, and also suggest that serotonin plays a prominent role in regulating the neuronal cyto-architecture in addition to its classical role as neurotransmitter.