The dentate gyrus of the ventral hippocampus (vDG) is strongly implicated in the pathophysiology of anxiety disorders. vDG is heavily innervated by the nucleus incertus (NI), a dorsal pontine locus highly sensitive to neurogenic stress and the main source of the neuropeptide, relaxin-3 (RLN3) in the rat brain. RLN3 receptor (RXFP3) activation has been shown to modulate behaviours, including stress-related anxiety. However, the effect of RLN3/RXFP3 signaling on vDG neuronal activity has not been examined. Therefore, this study investigated the influence of RLN3 on rat vDG neuronal activity ex vivo, and assessed the related anatomical substrates. Whole-cell, patch-clamp recordings in horizontal brain slices, revealed that RLN3 (100 nM) decreased vDG granule cell excitability. Viral-based, neural tract-tracing revealed that the hilus and the inner molecular layer of vDG were similarly innervated by NI-originating and RLN3-positive nerve fibers. The presence of RLN3 in NI-originating fibers, indicated that NI is a major source of this neuropeptide within vDG. Multiplex, fluorescence in situ hybridization revealed that vDG hilar interneurons, but not granule cells, express RXFP3 mRNA.Our findings indicate that the NI-originating, RLN3 innervation of vDG can modulate vDG activity via direct activation of RXFP3 on hilar interneurons, resulting in altered granule cell excitability. These effects of RLN3/RXFP3 signaling in the vDG are likely to contribute to the regulation of stress and anxiety-related behaviors. Funding: National Science Centre, Poland grants UMO-2018/30/E/NZ4/00687 and UMO-2023/49/B/NZ4/01885 and MiniGrant2023-ID.UJ.