BACKGROUND: Stimulation of the vagus nerve via implanted electrodes is currently used to treat refractory epilepsy and depression. Recently, a non-invasive approach to vagal stimulation has demonstrated similar beneficial effects, but it remains unclear whether these effects are mediated via activation of afferent vagal fibers. OBJECTIVE: The present study was designed to ascertain whether afferent vagal projections can be accessed non-invasively by transcutaneous electrical stimulation of the antero-lateral surface of the neck, which overlies the course of the vagus nerve. METHODS: Thirteen healthy subjects underwent 2 fMRI scans in one session. Transcutaneous electrical stimulation was applied for 2 min to the right postero-lateral surface of the neck during scan #1 (control condition, sternocleidomastoid stimulation: "SCM") and to the right antero-lateral surface of the neck during scan #2 (experimental condition, non-invasive vagus nerve stimulation: "nVNS"). Two analyses were conducted using FSL (whole-brain and brainstem; corrected, p < 0.01) to determine whether nVNS activated vagal projections in the brainstem and forebrain, compared to baseline and SCM stimulation. RESULTS: Compared to baseline and control (SCM) stimulation, nVNS significantly activated primary vagal projections including: nucleus of the solitary tract (primary central relay of vagal afferents), parabrachial area, primary sensory cortex, and insula. Regions of the basal ganglia and frontal cortex were also significantly activated. Deactivations were found in the hippocampus, visual cortex, and spinal trigeminal nucleus. CONCLUSION: The present findings provide evidence in humans that cervical vagal afferents can be accessed non-invasively via transcutaneous electrical stimulation of the antero-lateral surface of the neck, which overlies the course of the nerve, suggesting an alternative and feasible method of stimulating vagal afferents.