The dopamine transporter (DAT) is an important regulator of brain dopamine (DA) homeostasis, controlling the intensity and duration of DA signaling. DAT is the target for psychostimulants - like cocaine and amphetamine - and plays an important role in neuropsychiatric disorders, including attention-deficit hyperactivity disorder and drug addiction. Thus, a thorough understanding of the mechanisms that regulate DAT function is necessary for the development of clinical interventions to treat DA-related brain disorders. Previous studies have revealed a plethora of protein-protein interactions influencing DAT cellular localization and activity, suggesting that the fine-tuning of DA homeostasis involves multiple mechanisms. We recently reported that G-protein beta-gamma (Gβγ 3) subunits bind directly to DAT and decrease DA clearance. Here we show that Gβγ 3 induces the release of DA through DAT. Specifically, a Gβγ 3-binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous cells and primary dopaminergic neurons in culture. Addition of the Gβγ 3 inhibitor gallein or DAT inhibitors prevents this effect. Residues 582 to 596 in the DAT carboxy terminus were identified as the primary binding site of Gβγ 3. A TAT peptide containing the Gβγ 3-interacting domain of DAT blocked the ability of mSIRK to induce DA efflux, consistent with a direct interaction of Gβγ 3 with the transporter. Finally, activation of a G-protein-coupled receptor, the muscarinic M5R, results in DAT-mediated DA efflux through a Gβγ 3-dependent mechanism. Collectively, our data show that Gβγ 3 interacts with DAT to promote DA efflux. This novel mechanism may have important implications in the regulation of brain DA homeostasis.