Introduction: Urotensin II (UII) is a potent vasoactive peptide that exerts differential effects on heart failure (HF) patients compared to health controls. However, the mechanism of action remains unclear. The role of soluble epoxide hydrolase (sEH) as a mediator of UII in the vasculature has not been explored. Aims: The aim of this study was to examine the effect of UII in the presence and absence of sEH inhibitor AUDA on skin microvessel tone in HF patients and healthy controls using iontophoresis and laser Doppler velocimetry. UII (10−7 M) and AUDA (10−10, 10−7, and 10−5 M) were administered to the forearm of participants by iontophoresis for 30 seconds. Laser Doppler velocimetry was performed for 5 minutes to measure flux through the subcutaneous blood vessels. Response (flux) was measured for 5 minutes per concentration with 25 continuous scans. Results: UII increased flux in healthy controls by 39% (P < 0.05) and increased flux in HF patients by 6% (ns). AUDA (10−10 and 10−7 M) administration further decreased flux by 115% (P < 0.05) and 255% (P < 0.0001), respectively in healthy controls. In HF patients, AUDA (10−10, 10−7, and 10−5 M) further increased flux by 77% (P < 0.05), 67% (P < 0.01), and 100% (P < 0.05), respectively. AUDA alone at 10−7 M increased flux in both groups by 31% (healthy controls, P < 0.05) and 36% (HF, P < 0.01). Conclusion: Taken together, the presence of HF appeared to abrogate the vasodilator responsiveness of sEH inhibitor. These results suggest an important role for both UII and sEH in vascular regulation and that sEH may be involved in mediating UII effects. Furthermore, the study highlights the therapeutic potential of sEH inhibitors for the treatment of HF.