The oxytocin (OXT) and relaxin-3 (RLN3) signalling systems are involved in shaping social and stress-related behaviours, but the interplay of these neural networks in relevant brain regions including the ventral hippocampus dentate gyrus (vDG), is under explored. The rat vDG is strongly innervated by OXT and RLN3-containing nerve fibres, but possible interactions of these neuropeptide systems in vDG are unknown. Therefore, we investigated the influence of OXT and RLN3 signalling, separately and in combination, on rat and human vDG neurons. Multiplex in situ hybridization (ISH) identified a moderate level of RLN3 receptor (RXFP3) and OXT receptor (OXTR) mRNA co-expression in vGAT1 mRNA-positive (GABA) neurons in rat and human vDG. Multi-electrode recordings from rat brain slices revealed a rapid, direct, yet opposing, influence of OXT and RLN3 on the activity of vDG neurons, with OXT activating and RLN3 inhibiting vDG neurons. Notably, the effect of each neuropeptide on vDG activity was diminished in the presence of the other neuropeptide. Application of RXFP3 and OXTR agonists (A2 and TGOT, respectively) in the presence of a KCNQ potassium channel blocker (XE991) attenuated the responses to the agonist alone. ISH also revealed the co-expression of RXFP3, OXTR and KCNQ2 mRNAs in rat vDG neurons. Our findings identify a novel, opposing interplay of OXT/OXTR and RLN3/RXFP3 signalling on vDG neuronal activity, involving KCNQ channels, which is likely involved in shaping social and stress-related behaviours in rodents and humans. Funding: National Science Centre, Poland: UMO-2023/49/B/NZ4/01885, UMO-2018/30/E/NZ4/00687; MiniGrant2023 ID.UJ; BrightFocus Foundation-A2021006; Alzheimer’s Association-AARFD-22-972099.