Background: Sex hormones influence vascular function. Whether boys with hypospadias who have insufficient androgen exposure during the masculinisation programming window have altered vascular function is unknown. Objective: To investigate whether vascular function is impaired in boys with hypospadias and to explore the putative role of eNOS. Methods: Peripheral arteries from excess foreskin tissue obtained from boys undergoing hypospadias repair (cases) or circumcision (controls) were used. Vascular function was assessed by myography. mRNA expression was measured by qPCR in vascular smooth muscle cells (VSMCs). Nitric oxide (NO) was measured by DAF fluorescence assay and peroxynitrite levels measured via ELISA. Results: 23 boys with hypospadias and 34 age-matched controls were studied. There were 18 (52%) cases of distal, 7 (22%) of midshaft and 9 (26%) of proximal hypospadias and none of them had biochemical evidence of hypogonadism or a variant in AR. Clinical cardiometabolic parameters were similar between groups. Endothelium-dependent relaxation to acetylcholine (ACh) and endothelium-independent relaxation to sodium nitroprusside (SNP) were reduced in arteries from cases vs controls (Emax %U46619: 72.4 vs 1.2, p<0.0001 and Emax %U46619: (42.7 vs 11.8, p<0.01 respectively). Incubation with the NO synthase inhibitor, L-NAME (1x10-5 M) worsened endothelial-dependent relaxation in controls (Emax % U46619: 76.8 vs 1.2, p<0.0001) but had no effect in cases (Emax % U46619:60.6 vs 72.4, p=0.3). Testosterone (1x10-7 M) ameliorated vascular relaxation (p<0.05), whereas17[[Unsupported Character - Symbol Font 𝝱]];-estradiol stimulation (1x10-9 M) did not. In cultured VSMCs, mRNA expression of eNOS and iNOS was reduced whereas that of nNOS was increased in cases versus controls. Nitric oxide production was reduced in cases (5 fold, p<0.01), as was peroxynitrite production (0.5 fold, p<0.05). Testosterone increased expression of eNOS in VSMCs. There was no difference in mRNA expression of the AR and GPRC6A but cases had increased expression of ESR1 (2.71 fold), ESR2 (2.63 fold) and GPR30 (2.86 fold) (p<0.05). Conclusion: Arteries in eugonadal boys with hypospadias show vascular dysfunction which involves impaired NOS/NO regulation effects that are ameliorated with testosterone but not oestrogen. These processes may predispose to long-term cardiovascular disease.