Dopamine (DA) homeostasis is essential for a variety of brain activities.Dopamine transporter (DAT)-mediated DA reuptake is one of the most critical mechanisms for the control of normal DA homeostasis.However, the molecular mechanisms underlying the regulation of DAT activity in the brain remain poorly understood.Here, we showed that a Rho family GEF protein Vav2 controls DAT cell surface expression and transporter activity through internalization of a protein complex, consisting of Vav2, glial cell linederived neurotrophic factor cognate receptor Ret and DAT.Mice deficient in either Vav2 or Ret displayed an elevation of DAT activity, resulting in a significant increase in the intracellular DA levels selectively in the nucleus accumbens.Vav2-/-mice exposed to cocaine showed reduced DAT activity and diminished behavioral cocaine response.Our data identify Vav2 as a critical regulator of DAT trafficking in vivo and a determinant of DA homeostasis in limbic DA neuron terminals.