Background Numerous protocols exist for isolating aortic endothelial and smooth muscle cells from small animals. However, establishing a protocol for isolating pure cell populations from large animal vessels that are more elastic has been challenging. We developed a simple sequential enzymatic approach to isolate highly purified populations of porcine aortic endothelial and smooth muscle cells. Methods The lumen of a porcine aorta was filled with 25 U/mL dispase solution and incubated at 37°C to dissociate the endothelial cells. The smooth muscle cells were isolated by mincing the tunica media of the treated aorta and incubating the pieces in 0.2% and then 0.1% collagenase type I solution. Results The isolated endothelial cells stained positive for von Willebrand factor, and 97.2% of them expressed CD31. Early‐ and late‐passage endothelial cells had a population doubling time of 38 hours and maintained a capacity to take up DiI‐Ac‐LDL and form tubes in Matrigel®. The isolated smooth muscle cells stained highly positive for alpha‐smooth muscle actin, and an impurities assessment showed that only 1.8% were endothelial cells. Population doubling time for the smooth muscle cells was ∼70 hours at passages 3 and 7; and the cells positively responded to endothelin‐1, as shown by a 66% increase in the intracellular calcium level. Conclusions This simple protocol allows for the isolation of highly pure populations of endothelial and smooth muscle cells from porcine aorta that can survive continued passage in culture without losing functionality or becoming overgrown by fibroblasts. This article is protected by copyright. All rights reserved