Neuropathic pain affects millions of people causing disability and impairing quality of life. Commonly used analgesics are generally characterized by limited therapeutic outcomes. The serotonin 5-HT1A receptor and the α2 adrenergic receptors are involved in central nociceptive mechanisms with a pivotal role in the inhibitory descending pain pathway. Since their stimulation may modulate the nervous signaling altered by neuropathies, the purpose of the present research is the study of the combined activation of 5-HT1A and α2 receptors by rationally designed imidazoline ligands ((S)-(-)-1 and 2–5) in a rat model of neuropathic pain (chronic constriction injury - CCI). On day 14 after nerve damage, the acute administration per os (p.o.) of low doses of (S)-(-)-1 (0.1–1 mg/kg) was able to significantly increase the pain threshold to mechanical noxious stimuli for more than 1 h. (S)-(-)-1 efficacy was confirmed by the decrease of spontaneous pain evaluated as hind limb weight bearing alterations. The clinically-used compound gabapentin (100 mg/kg p.o.) induced a pain relieving effect similar to (S)-(-)-1 administered at 100 fold lower dose. In the same model, the selected analogues, compounds 2–5 (1 mg/kg p.o.) were effective 30 min after administration. In particular, 5 fully reverted the CCI-induced hypersensitivity. The pain relieving activity of 5 was significantly prevented by the selective 5-HT1A receptor antagonist WAY 100635 (1 mg/kg intraperitoneally, i.p.) and, at a lesser extent, by the α2 antagonist yohimbine (3 mg/kg i.p.). A novel pharmacodynamic approach to the treatment of neuropathic pain is presented.