Background: Substance P (SP) is a peptide neurotransmitter in many central and peripheral neural pathways. Its precise role in human physiology has been difficult to elucidate. We used the selective neurokinin 1 (NK1) antagonist aprepitant as a pharmacologic probe to determine the role of endogenous SP in human cardiovascular regulation. Methods and Results: We performed a randomized, double blind, placebo controlled, crossover trial in healthy subjects. Blockade of endogenous NK₁ receptors reduced resting muscle sympathetic activity 38% (p=0.002), reduced systemic vascular resistance by 25% (p=0.021), and increased cardiac index by 47% (p=0.006). This constellation of changes did not however alter either blood pressure or heart rate in the supine position. NK₁ antagonism also raised orthostatic heart rate change by 38% (p=0.023), though during the incremental postural adjustment on the tilt table neither heart rate nor blood pressure was altered significantly. Despite a mildly attenuated vagal baroreflex with SP blockade, the depressor and pressor responses to nitroprusside and to phenylephrine did not differ compared to placebo suggesting other compensatory mechanisms. Conclusions: NK₁ blockade manifests as a decrease in muscle sympathetic nerve activity and systemic vascular resistance. Our study suggests substance P exerts a tonic enhancement of sympathetic outflow to some cardiovascular structures via its modulation of the NK₁ receptor. Most likely, this ubiquitous neurotransmitter exerts effects at multiple sites that, in the aggregate, are relatively well-compensated under many circumstances, but which may emerge with perturbations. This study is consistent with a role for Substance P afferents in supporting peripheral vascular resistance.