Introduction: A strong association between cardiovascular disease and obesity has been reported. However, the role of adiponectin in the effect of physical activity on mesenteric circulation and its underlying mechanism has been unknown. We investigated the protective effect of voluntary wheel running (EX) on endothelial function in the mesenteric artery of obese mice and its underlying mechanisms. Methods: C57BL/6J male mice were randomly divided into four groups: (1) control low-fat diet (LF), (2) LF diet with free access to a voluntary running wheel (LF-EX), (3) high-fat diet (HF; 45% of calories from fat), and (4) HF-EX. The protein expressions of total-endothelial nitric oxide synthase (t-eNOS), adiponectin receptors 1 and 2, AMPK, Ob (leptin), inflammatory markers, and proteins regulating apoptosis were quantified by Western blot analysis. Statistical comparisons between groups were performed with two-way ANOVA with Fisher’s LSD test. Results: The protein expressions of t-eNOS, adiponectin receptors 1 and 2, and p-AMPK in HF were lower compared to LF, but EX increased the protein levels ( P < 0.05). Ob in HF was higher compared to LF, but EX decreased it ( P < 0.05). IL-1β, TNF-α, and Caspase-1 in HF were higher compared to LF, but EX decreased the protein levels ( P < 0.05). There was no difference in expressions of AMPK, CHOP, Bcl-2, and GADD153 between groups. Conclusion: Voluntary wheel running alleviates e-NOS-mediated endothelial dysfunction in the mesenteric artery of obese mice possibly via increased adiponectin receptors and decreased inflammation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.