Accurate discrimination between new and familiar stimuli is essential for adaptive responses to an ever-changing environment, and disruptions in this process are strongly related to various neuropsychiatric disorders. Stress is a primary mediator of the development of novelty response-related deficiencies; therefore, we investigated the neuronal mechanisms underlying the possible control of the interpeduncular nucleus (IPN), implicated in signaling novelty preference, by the highly stress-sensitive, brainstem nucleus incertus (NI), the primary central source of the neuropeptide, relaxin-3 (RLN3).Multiplex in situ hybridization revealed that RLN3 receptor (RXFP3) mRNA, was expressed by dopaminergic D1 and cholinergic α5 receptor mRNA-expressing IPN neurons, implicated in novelty and familiarity signaling in IPN, respectively. Patch-clamp electrophysiological recordings of IPN neuron activity combined with optogenetic stimulation of NI-originating fibers revealed a direct, monosynaptic inhibitory innervation of IPN by NI in male rats. Results of viral-based, neural tract-tracing revealed that NI neurons innervating the IPN, project to several other brain regions involved in the control of the stress and novelty related responses, including the medial septum and ventral hippocampus. Behavioral studies demonstrated that chemogenetic activation of the NI-IPN projection produced increased exploration time and reduced grooming behavior.Together, our findings suggest the NI is involved in stress-related control of novelty preference and related behaviors. Importantly, the results of the current study provide valuable insights into the neuronal mechanisms that may underlie stress-related neuropsychiatric diseases associated with impaired novelty preference.Funding: National Science Centre Poland UMO-2018/30/E/NZ4/00687, UMO-2023/49/B/NZ4/01885; Jagiellonian University U1U/W18/NO/28.40.