Long-term potentiation (LTP) of synaptic strength in nociceptive pathways is a cellular model of hyperalgesia. The emerging literature suggests a role for cytokines released by spinal glial cells for both LTP and hyperalgesia. However, the underlying mechanisms are still not fully understood. In rat lumbar spinal cord slices, we now demonstrate that conditioning high-frequency stimulation of primary afferents activated spinal microglia within <30 min and spinal astrocytes within ∼2 s. Activation of spinal glia was indispensible for LTP induction at C-fiber synapses with spinal lamina I neurons. The cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), which are both released by activated glial cells, were individually sufficient and necessary for LTP induction via redundant pathways. They differentially amplified 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)-propanoic acid receptor-mediated and N-methyl-d-aspartic acid receptor-mediated synaptic currents in lamina I neurons. Unexpectedly, the synaptic effects by IL-1β and TNF-α were not mediated directly via activation of neuronal cytokine receptors, but rather, indirectly via IL-1 receptors and TNF receptors being expressed on glial cells in superficial spinal dorsal horn. Bath application of IL-1β or TNF-α led to the release profiles of pro-inflammatory and anti-inflammatory cytokines, chemokines, and growth factors, which overlapped only partially. Heat hyperalgesia induced by spinal application of either IL-1β or TNF-α in naive animals also required activation of spinal glial cells. These results reveal a novel, decisive role of spinal glial cells for the synaptic effects of IL-1β and TNF-α and for some forms of hyperalgesia.