Background. The goals of this study were: (1) to develop a large animal model to study endothelial function, and (2) to determine if arterial thrombosis induces endothelial dysfunction in vivo. Methods. Surgical exposure of the porcine iliac and femoral arteries was performed. Normal porcine arteries were compared with arteries subjected to 90 min of arterial thrombosis. External iliac artery (EIA) luminal diameters were measured using M- and B-mode duplex ultrasound. Endothelium-dependent relaxation (EDR) and endothelium-independent relaxation (EIR) were measured using acetylcholine (ACh) and sodium nitroprusside (NTP), respectively. Endothelial integrity was determined by factor VIII immunohistochemistry (F8) and scanning electron microscopy (SEM). Nitric oxide levels were determined using a chemiluminescence assay of nitrite/nitrate metabolites (NOx). Continuous variables were analyzed using the two-tailed Student t test. Results. Control artery EDR was 80 ± 7.1% (± SE), while arteries exposed to luminal thrombus for 90 min had an EDR of 55.2 ± 5.7% (ACh = 15 μg/min, n = 11, P = 0.0231). EIR was preserved in normal and thrombosis groups with uniform response to NTP (4.92 ± 0.1 cm vs 5.07 ± 0.42 cm, P = 0.76). F8 staining identified endothelium in all groups. SEM analysis revealed an intact monolayer of endothelium after thrombosis. Local NOx levels were 17.3% lower after 90 min of thrombosis (49.3 μM vs 40.8 μM, n = 16, P < 0.001). Conclusions. Luminal thrombus induces arterial dysfunction acutely without causing endothelial cell loss. EIR remains unaffected, indicating normal smooth muscle cell function. NOx levels suggest that nitric oxide levels are decreased acutely after thrombosis. The development of this porcine large animal model allows the in vivo study of vasospasm and alternative thrombolytic regimens.