Antidepressants, such as duloxetine and amitriptyline, are effective for treating patients with chronic neuropathic pain. Inhibiting norepinephrine and serotonin transporters at presynaptic terminals raises extracellular concentrations of norepinephrine. The α1- and α2-adrenergic receptor agonists inhibit glutamatergic input from primary afferent nerves to the spinal dorsal horn. However, the contribution of spinal α1- and α2-adrenergic receptors to the analgesic effect of antidepressants and associated synaptic plasticity remains uncertain. In this study, we showed that systemic administration of duloxetine or amitriptyline acutely reduced tactile allodynia and mechanical and thermal hyperalgesia caused by spinal nerve ligation in rats. In contrast, duloxetine or amitriptyline had no effect on nociception in sham rats. Blocking α1-adrenergic receptors with WB-4101 or α2-adrenergic receptors with yohimbine at the spinal level diminished the analgesic effect of systemically administered duloxetine and amitriptyline. Furthermore, intrathecal injection of duloxetine or amitriptyline similarly attenuated pain hypersensitivity in nerve-injured rats; the analgesic effect was abolished by intrathecal pretreatment with both WB-4101 and yohimbine. In addition, whole-cell patch-clamp recordings in spinal cord slices showed that duloxetine or amitriptyline rapidly inhibited dorsal root-evoked excitatory postsynaptic currents in dorsal horn neurons in nerve-injured rats but had no such effect in sham rats. The inhibitory effect of duloxetine and amitriptyline was abolished by the WB-4101 and yohimbine combination. Therefore, antidepressants attenuate neuropathic pain predominantly by inhibiting primary afferent input to the spinal cord via activating both α1- and α2-adrenergic receptors. This information helps the design of new strategies to improve the treatment of neuropathic pain.