The interpeduncular nucleus (IPN) is a highly complex and evolutionarily conserved midbrain locus involved in social novelty preference and anxiety control. The IPN is characterised by a high density of the nerve-growth factor (NGF) receptor, TrkA; and notably, social stress strongly promotes brain NGF synthesis. The IPN is densely innervated by the nucleus incertus (NI), a stress-sensitive region that is the primary source of the neuropeptide, relaxin-3 (RLN3). In turn, IPN innervates the ventral hippocampus (vHPC), another key locus for social behaviour and anxiety control. However, the nature of NI-IPN-vHPC axis function is unknown.Multiplex in situ hybridization revealed co-expression of TrkA and RLN3 receptor (RXFP3) mRNA in GABAergic IPN neurons. Viral-based, neural tract-tracing revealed an extensive RLN3 innervation of the IPN originating in the NI. Multielectrode array recordings revealed excitatory and inhibitory effects of the RXFP3-selective agonist, A2, on IPN neuronal activity, and A2-evoked outward currents were observed in whole-cell, patch-clamp recordings. Additional neural tract-tracing studies revealed a dense, ipsilateral innervation of vHPC by the rostral and lateral IPN nuclei, and these areas were the most densely innervated by the NI.Our findings indicate that NGF-sensitive, IPN neurons are influenced by the NI RLN3/RXFP3 system, and that the IPN nuclei under NI control, directly innervate the vHPC. Together, our data describe the key neuronal substrates underlying the function of the NI-IPN-vHPC axis, implicated in the control of social interaction and anxiety.Funding: National Science Centre, Poland (UMO-2018/30/E/NZ4/00687; UMO 2021/41/N/NZ4/04499; UMO-2023/49/B/NZ4/01885).