The growing application of transcranial direct current stimulation (tDCS) in both experimental and clinical settings prompts an exploration of its underlying neurobiological mechanisms. Emerging evidence suggests that tDCS indirectly modulates brain activity via cranial nerve pathways, notably the trigeminal nerve. These pathways play a role in controlling neurotransmitter release and possible upstream effects. However, the electrophysiological effects of direct current stimulation on the trigeminal nerve (DC-TNS) within brainstem nuclei remain uncharted territory.Hereby, DC-TNS (± 0.25- ± 3 mA) impact on the firing rates was examined in the principal sensory (NVsnpr) and the mesencephalic nuclei (MeV), the dorsal and median raphe nuclei (DRN and MnRN), and the locus coeruleus (LC) using experiments involving 41 male Sprague Dawley rats (n=10 for each nucleus; except for DRN and MnRN that were subsequently examined in the same rat) under urethane anesthesia. Single-unit recordings were obtained through a 32-channel silicon probe during three one-minute intervals: before, during, and after stimulation. Xylocaine block of the trigeminal nerve served as a control.DC-TNS increased neuronal spiking activity in NVsnpr, MeV, DRN, and LC, but not MnRN, with a subsequent return to baseline post-stimulation. No difference was found between anodic and cathodic stimulation. Notably, DC-TNS at 3 mA on the blocked trigeminal nerve did not increase firing rates. These findings suggest that tDCS effects may be at least partially influenced by the transcutaneous route and TNS. This investigation holds significant translational importance, providing detailed mechanistic insights crucial for refining the implementation of tDCS in various pathophysiological conditions.