HIV-1 infection of the nervous system causes various neurological diseases, and synaptic degeneration is likely a critical step in the neuropathogenesis. Our prior studies revealed a significant decrease of synaptic protein, specifically in the spinal dorsal horn of patients with HIV-1 in whom pain developed, suggesting a potential contribution of synaptic degeneration to the pathogenesis of HIV-associated pain. However, the mechanism by which HIV-1 causes the spinal synaptic degeneration is unclear. Here, we identified a critical role of microglia in the synaptic degeneration. In primary cortical cultures (day in vitro 14) and spinal cords of 3- to 5-month-old mice (both sexes), microglial ablation inhibited gp120-induced synapse decrease. Fractalkine (FKN), a microglia activation chemokine specifically expressed in neurons, was upregulated by gp120, and knockout of the FKN receptor CX3CR1, which is predominantly expressed in microglia, protected synapses from gp120-induced toxicity. These results indicate that the neuron-to-microglia intercellular FKN/CX3CR1 signaling plays a role in gp120-induced synaptic degeneration. To elucidate the mechanism controlling this intercellular signaling, we tested the role of the Wnt/β-catenin pathway in regulating FKN expression. Inhibition of Wnt/β-catenin signaling blocked both gp120-induced FKN upregulation and synaptic degeneration, and gp120 stimulated Wnt/β-catenin-regulated FKN expression via NMDA receptors (NMDARs). Furthermore, NMDAR antagonist APV, Wnt/β-catenin signaling suppressor DKK1, or knockout of CX3CR1 alleviated gp120-induced mechanical allodynia in mice, suggesting a critical contribution of the Wnt/β-catenin/FKN/CX3R1 pathway to gp120-induced pain. These findings collectively suggest that HIV-1 gp120 induces synaptic degeneration in the spinal pain neural circuit by activating microglia via Wnt3a/β-catenin-regulated FKN expression in neurons.SIGNIFICANCE STATEMENT Synaptic degeneration develops in the spinal cord dorsal horn of HIV patients with chronic pain, but the patients without the pain disorder do not show this neuropathology, indicating a pathogenic contribution of the synaptic degeneration to the development of HIV-associated pain. However, the mechanism underlying the synaptic degeneration is unclear. We report here that HIV-1 gp120, a neurotoxic protein that is specifically associated with the manifestation of pain in HIV patients, induces synapse loss via microglia. Further studies elucidate that gp120 activates microglia by stimulating Wnt/β-catenin-regulated fractalkine in neuron. The results demonstrate a critical role of microglia in the pathogenesis of HIV-associated synaptic degeneration in the spinal pain neural circuit.