AIMS: Ventricular arrhythmias are associated with sympathoexcitation and increased co-transmitter neuropeptide Y (NPY) levels. Vagal nerve stimulation (VNS) has been reported to decrease release of norepinephrine, while NPY has been reported to decrease acetylcholine release ex vivo by binding Y2 receptors on parasympathetic nerves. We hypothesized that VNS reduces NPY levels via a muscarinic receptor (MR) mediated mechanism in vivo and that, in turn, blockade of pre-synaptic Y2R can further enhance the effects of VNS and decrease the effects of sympathoexcitation by increasing vagal tone. METHODS AND RESULTS: Single-cell RNA sequencing of rat stellate ganglia and immunohistochemistry were performed and identified the M2 receptor as the predominant subtype on NPY-expressing sympathetic neurons. Ex vivo field stimulation of rat stellate ganglia, before and after application of carbamylcholine (CCH; muscarinic agonist) and atropine (muscarinic blocker) showed that CCH reduced NPY release, while the addition of atropine increased NPY levels. Subsequently, to validate ex vivo findings, in vivo effects of VNS during bilateral stellate ganglia stimulation (BSS) on NPY release with and without atropine were evaluated and haemodynamic and electrophysiological parameters, including ventricular activation recovery intervals (ARIs, a surrogate for action potential duration), and real-time in vivo interstitial NPY levels were measured. Post-atropine, suppression of NPY by VNS was significantly diminished, confirming a MR mediated mechanism in vivo. Finally, in a porcine model in vivo, effects of VNS on NPY levels and of the Y2R blocker, BIIE0246, during BSS were tested. These studies demonstrated that Y2R blockade significantly reduced the cardiac effects of BSS on systolic pressure, inotropy, and ARIs. While the ventricular effects of VNS, including suppression of interstitial NPY levels, haemodynamic, and electrophysiological parameters were enhanced by Y2R blockade, heart rate remained unaffected. CONCLUSION: Vagal activation reduces interstitial NPY levels via a pre-synaptic sympathetic M2R mechanism. Y2R inhibition reduces effects of sympathoexcitation and enhances the effects of VNS in vivo. These findings highlight the role of NPY in sympathovagal crosstalk and suggest Y2R as a potential target to modulate autonomic balance.