Key points ‘Functional sympatholysis’ describes the ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction, and is critical to ensure proper blood flow and oxygen delivery to metabolically active skeletal muscle. The signalling mechanism responsible for sympatholysis in healthy humans is unknown. Evidence from animal models has identified endothelium‐derived hyperpolarization (EDH) as a potential mechanism capable of attenuating sympathetic vasoconstriction. In this study, increasing endothelium‐dependent signalling during exercise significantly enhanced the ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction in humans. This is the first study in humans to identify endothelium‐dependent regulation of sympathetic vasoconstriction in contracting skeletal muscle, and specifically supports a role for EDH‐like vasodilatory signalling. Impaired functional sympatholysis is a common feature of cardiovascular ageing, hypertension and heart failure, and thus identifying fundamental mechanisms responsible for sympatholysis is clinically relevant. Abstract Stimulation of α‐adrenoceptors elicits vasoconstriction in resting skeletal muscle that is blunted during exercise in an intensity‐dependent manner. In humans, the underlying mechanisms remain unclear. We tested the hypothesis that stimulating endothelium‐dependent vasodilatory signalling will enhance the ability of contracting skeletal muscle to blunt α1‐adrenergic vasoconstriction. Changes in forearm vascular conductance (FVC; Doppler ultrasound, brachial intra‐arterial pressure via catheter) to local intra‐arterial infusion of phenylephrine (PE; α1‐adrenoceptor agonist) were calculated during (1) infusion of the endothelium‐dependent vasodilators acetylcholine (ACh) and adenosine triphosphate (ATP), the endothelium‐independent vasodilator (sodium nitroprusside, SNP), or potassium chloride (KCl) at rest; (2) mild or moderate intensity handgrip exercise; and (3) combined mild exercise + ACh, ATP, SNP, or KCl infusions in healthy adults. Robust vasoconstriction to PE was observed during vasodilator infusion alone and mild exercise, and this was blunted during moderate intensity exercise (ΔFVC: −34 ± 4 and −34 ± 3 vs. −13 ± 2%, respectively, P < 0.05). Infusion of ACh or ATP during mild exercise significantly attenuated PE vasoconstriction similar to levels observed during moderate exercise (ACh: −3 ± 4; ATP: −18 ± 4%). In contrast, infusion of SNP or KCl during mild exercise did not attenuate PE‐mediated vasoconstriction (−32 ± 5 and −46 ± 3%). To further study the role of endothelium‐dependent hyperpolarization (EDH), ACh trials were repeated with combined nitric oxide synthase and cyclooxygenase inhibition. Here, PE‐mediated vasoconstriction was blunted at rest (blockade: −20 ± 5 vs. control: −31 ± 3% vs.; P < 0.05) and remained blunted during exercise (blockade: −15 ± 5 vs. control: −14 ± 5%). We conclude that stimulation of EDH‐like vasodilatation can blunt α1‐adrenergic vasoconstriction in contracting skeletal muscle of humans.