Over 100 million Americans experience some forms of physical pain, a quarter of whom struggle daily to cope with chronic and persistent pain conditions. One of the risks associated with existing analgesic drugs, beyond some manageable side effects, is the potential for drug dependence and abuse. Glycinergic inhibition plays a pivotal role in spinal nociception. Enhancing glycine receptor (GlyR) activities by positive allosteric modulators (PAMs) has been recognized as a promising alternative to opioids for treating chronic pain. By combining structural biology, electrophysiology, and in vivo studies, we discovered a novel drug binding site in the human GlyRs that mediates marijuana’s analgesic action independent of its psychoactive side effects. We further discovered a new molecular scaffold that potentiates GlyRs with little cross reactivity with opioid receptors and other psychotropic receptors. A lead drug candidate from this scaffold was found to be specific positive allosteric modulators for α3-containing GlyRs and have higher potencies than morphine in suppressing neuropathic and inflammatory pain in rodents. Using RNAscope for in situ hybridization, we also discovered abundant colocalization of α3GlyR with a special group of projection neurons in the midbrain. These intriguing findings led us to hypothesize that α3GlyRs in the superficial layer of the spinal dorsal horn and in the midbrain play a dual role as important targets for analgesia and as inhibitory regulators for the reward circuits, respectively. We have collected ample preliminary data to support the following three specific aims: Aim 1: Investigate the role of spinal α3GlyR as an effective molecular target to alleviate mechanical and thermal hypersensitivity in neuropathic and inflammatory pain; Aim 2: Understand α3GlyR’s regulation of the midbrain projection neurons as a key cellular target for antinociception and anti-psychomotor stimulation; and Aim 3: Elucidate the coupling of glycinergic and dopaminergic signaling pathways to harness the dual action of selective positive allosteric modulation of α3GlyRs for both analgesia and reduction of reward-seeking behavior, focusing on drug-seeking and instrumental learning of self-administration tests. These mechanism-guided investigations will complement and further enhance the discovery of new α3GlyR-targeting drugs for safe and effective pain treatment.