This study aimed to delve into the role of the brainstem nucleus incertus (NI) as a structure involved in stress-response generation and its interactions with the ventral tegmental area (VTA), a key component of the brain's reward system. By tracing neural pathways using a retrograde viral vector, the NI-originating innervation of the VTA was confirmed within the midbrain of Sprague-Dawley rats. Immunohistochemical staining revealed that only a small percentage of NI neurons containing the relaxin-3 neuropeptide also project to the VTA, suggesting specialization of the connectivity between these regions. Behavioral experiments have revealed an increase in the activity of NI neurons, as evidenced by c-Fos expression, in response to painful stimuli. This observation underscores the role of the NI in processing adverse environmental conditions, thereby bolstering its significance in the generation of stress response. Furthermore, electrophysiological recordings shed light on the responses of NI neurons to painful stimuli, primarily revealing excitatory reactions. Intriguingly, the response direction of some neurons varied in correlation with alternating brain states, indicating dynamics in the information processing that takes part within the NI. Understanding the role of the NI in responding to stimuli of potentially stressogenic and aversive properties contributes to unraveling the neural mechanisms underlying stress and reward processing, thereby offering potential therapeutic targets for stress-related disorders. Funding: National Science Centre in Poland, PRELUDIUM 21, 2022/45/N/NZ4/03171