The study is to establish a novel method to determine the endothelial function in mouse carotid arteries in vivo by using high-resolution ultrasound images. Atherosclerosis in carotid arteries is induced in ApoE(-/-) mice with a Western diet. The ultrasound of the ventral neck generates clear pictures of the common carotid arteries. Acetylcholine at the range from 5 to 20 μg/kg/min (iv) is able to induce a dose-dependent relaxation as shown by the increased diameter of these normal mouse carotid arteries, which is impaired in atherosclerotic arteries. The endothelial function determined by ultrasound images in vivo matches well with that determined in isolated carotid arterial rings in vitro. All animals survived after the endothelial function measurement. In this study, we have established a standard method to determine the mouse endothelial function in vivo. It is a reliable, safe, and survival method that could be used repetitively in mouse arteries.