The role of glial cell line-derived neurotrophic factor (GDNF) in nociceptive pathways is still controversial, as both pronociceptive and antinociceptive actions have been reported. To elucidate this role in the mouse, we performed combined structural and functional studiesin vivoand in acute spinal cord slices where C-fiber activation was mimicked by capsaicin challenge.Nociceptors and their terminals in superficial dorsal horn (SDH; laminae I–II) constitute two separate subpopulations: the peptidergic CGRP/somatostatin+ cells expressing GDNF and the nonpeptidergic IB4+ neurons expressing the GFRα1-RET GDNF receptor complex. Ultrastructurally the dorsal part of inner lamina II (LII_id) harbors a mix of glomeruli that either display GDNF/somatostatin (GIb)-IR or GFRα1/IB4 labeling (GIa). LII_id thus represents the preferential site for ligand-receptor interactions.Functionally, endogenous GDNF released from peptidergic CGRP/somatostatin+ nociceptors upon capsaicin stimulation exert a tonic inhibitory control on the glutamate excitatory drive of SDH neurons as measured after ERK1/2 phosphorylation assay. Real-time Ca²⁺imaging and patch-clamp experiments with bath-applied GDNF (100 nm) confirm the presynaptic inhibition of SDH neurons after stimulation of capsaicin-sensitive, nociceptive primary afferent fibers. Accordingly, the reduction of the capsaicin-evoked [Ca²⁺]_irise and of the frequency of mEPSCs in SDH neurons is specifically abolished after enzymatic ablation of GFRα1. Therefore, GDNF released from peptidergic CGRP/somatostatin+ nociceptors acutely depresses neuronal transmission in SDH signaling to nonpeptidergic IB4+ nociceptors at glomeruli in LII_id. These observations are of potential pharmacological interest as they highlight a novel modality of cross talk between nociceptors that may be relevant for discrimination of pain modalities.