Affecting around 10% of world population, chronic pain and its related psychiatric comorbidities are major health issues. Implication of neuropeptides in modulation of pain remains poorly described in the brain. The relaxin-3 (RLN3) neuropeptide displays antidepressant and anxiolytic effects, and our preliminary results indicate an analgesic role in rodents. RLN3 is expressed by nucleus incertus (NI) neurons that project to different cortical (e.g. anterior cingulate cortex (ACC)) and subcortical (e.g. amygdala) areas of pain matrix.We aim at studying the pain modulatory effects of RLN3/RXFP3 system by using pharmacological, behavioral and anatomical approaches in a mouse model of persistent inflammatory pain.Persistent inflammation has been induced by injection of Complete Freund’s Adjuvant (CFA) in the paw of the animal. Intra-amygdalar injection of RXFP3 agonists alleviated both mechanical and thermal pain, while intra-ACC injection had an effect only on mechanical sensitization. The effect of AAV-mediated chronic release of another RXFP3 agonist (R3/I5) confirmed these effects in the ACC and amygdala. Tracing experiments (eGFP) of NI RLN3 neurons showed a dense but heterogenous network.In situ hybridization experiments demonstrated RXFP3 mRNA expression in somatostatin interneurons both in the ACC and amygdala, with an increase of RXFP3 expression in the ACC in pain condition. 3D quantification in the ACC indicated an increase in the number RLN3 profiles, but a decrease in their volume under inflammatory conditions.Our data highlight the plasticity of the RLN3/RXFP3 system and a novel antinociceptive role for this peptide family, suggesting its therapeutic potential in persistent pain conditions.