Endogenous nitric oxide (NO) plays important physiological roles in the body. As a small diatomic molecule, NO has been assumed to freely diffuse in tissues with a diffusion rate similar to that in water. However, this assumption has not been tested experimentally. In this study, a modified Clark-type NO electrode attached with a customized aorta holder was used to directly measure the flux of NO diffusion across the aortic wall at 37°C. Experiments were carefully designed for accurate measurements of the apparent NO diffusion coefficient D and the partition coefficient α in the aortic wall. A mathematical model was presented for analyzing experimental data. It was determined that α = 1.15 ± 0.11 and D = 848 ± 45 μm^2/s (n = 12). The NO diffusion coefficient in the aortic wall is nearly fourfold smaller than the reported diffusion coefficient in solution at 37°C, indicating that NO diffusion in the vascular wall is no longer free, but markedly dependent on the environment in the tissue where these NO molecules are. These results imply that the NO diffusion rate in the vascular wall may be upregulated and downregulated by certain physiological and/or pathophysiological processes affecting the composition of tissues.