In response to 5-hydroxytryptamine (5-HT), the type 1 serotonin receptors (5-HT1Rs) preferentially couple to the inhibitory G protein and elicit many physiological and behavioral processes. However, their regulation by intracellular protein kinases has not been fully investigated. In this study, we identified that glycogen synthase kinase-3 (GSK3) differentially regulates 5-HT1Rs. In receptor-expressing cells and brain slices, activation of both 5-HT1AR and 5-HT1BR reduced forskolin-stimulated cAMP production, but only the effect of 5-HT1BR was abolished by selective GSK3 inhibitors, deletion of GSK3 by RNAi, or overexpression of impaired GSK3 mutants (R96A and K85,86A). A consensus GSK3 phosphorylation sequence was identified between the serine-154 and threonine-158 in the second intracellular loop of 5-HT1BR. Mutation of either serine-154 or threonine-158 to alanine significantly reduced response of 5-HT1BR to 5-HT. Active GSK3 interacted with resting 5-HT1BR to form a protein complex. The interaction was enhanced by receptor activation, abolished by GSK3 inhibitors, and dependent on the phosphorylation state of serine-154. In addition, regulation of 5-HT1BR by GSK3 changed the dynamics of agonist-induced cell surface receptor internalization, in which lack of phosphorylation at Ser154 resulted in sustained reduction of 5-HT1BR at the cell surface. Although the physiological consequences of selective regulation of 5-HT1BR by GSK3 remain to be identified, findings in this study reveal a new function of GSK3 as a protein kinase that is able to selectively regulate G protein-coupled receptors.Most serotonin (5-HT) receptors are G protein-coupled receptors (GPCRs) that are classified by their sequence homology and by the type of G proteins and signal transduction pathways with which they are associated (Hannon and Hoyer, 2008). The type 1 5-HT receptors (5-HT1Rs), including subtypes A (5-HT1AR) and B (5-HT1BR), are mainly coupled to the inhibitory G protein, which links the receptors to inhibition of adenylyl cyclase and regulation of several other signal pathways (Raymond et al., 2001). 5-HT1AR and 5-HT1BR are found in several brain areas, such as the dorsal raphe nucleus, nucleus accumbens, cerebral cortex, hippocampus, and striatum. In the raphe nucleus, 5-HT1AR and 5-HT1BR are characterized as autoreceptors, with the former located at the somatodendritic area and the latter at the axon terminus. In many other brain regions and peripheral tissues, 5-HT1AR and 5-HT1BR also function as heteroreceptors in nonserotonergic neurons (Hannon and Hoyer, 2008). 5-HT1Rs are involved in many physiological processes in the brain, especially regulating turnover of 5-HT and other neurotransmitters in several brain regions (Ase et al., 2000). 5-HT1Rs are a major group of serotonin receptors that modulate behaviors, especially those relevant to neuropsychiatric diseases, such as anxiety, mood, and aggression (Gingrich and Hen, 2001). Therefore, proper regulation of activities of 5-HT1Rs is crucially important in main...