The neurobiological mechanisms of action underlying antidepressant drugs remain poorly understood. Desipramine (DMI) is an antidepressant classically characterized as an inhibitor of norepinephrine reuptake. Available evidence, however, suggests a mechanism more complex than simple reuptake inhibition. In the present study, we have characterized the direct interaction between DMI and the α(2A)-adrenergic receptor (α(2A)AR), a key regulator of noradrenergic neurotransmission with altered expression and function in depression. DMI alone was found to be sufficient to drive receptor internalization acutely and a robust down-regulation of α(2A)AR expression and signaling following prolonged stimulation in vitro. These effects are achieved through arrestin-biased regulation of the receptor, as DMI selectively induces recruitment of arrestin but not activation of heterotrimeric G proteins. Meanwhile, a physiologically relevant concentration of endogenous agonist (norepinephrine) was unable to sustain a down-regulation response. Prolonged in vivo administration of DMI resulted in significant down-regulation of synaptic α(2A)AR expression, a response that was lost in arrestin3-null animals. We contend that direct DMI-driven arrestin-mediated α(2A)AR down-regulation accounts for the therapeutically desirable but mechanistically unexplained adaptive alterations in receptor expression associated with this antidepressant. Our results provide novel insight into both the pharmacology of this antidepressant drug and the targeting of the α(2A)AR in depression.