Objective: Vascular dilation dysfunction has been linked with risk of cardiovascular disease. This study was undertaken to investigate the relationship between physical activity and vascular dilation function in healthy middle-aged adults to help explaining the effect of physical activity on preventing cardiovascular disease. Method: We recruited 91 healthy middle-aged adults to complete a serf-report 7-day physical activity recall questionnaire and an exam of brachial artery flow-mediated dilation(FMD) and Nitroglycerin-mediated dilation (NMD) detected by ultrasound. The relationship between physical activity level (PAL) and FMD and NMD were explored. Result: Physical activity showed a significant and positive relationship with the brachial artery FMD, even after adjustment for possible confounding factors (r=0.363, P<0.01). The group of high physical activity level had the highest FMD. The means of FMD (%) in low, moderate and high PAL groups were respectively 8.70%±3.93%,9.01%±3.22% and 12.38±5.67% with significant difference between individuals of low and high PAL and between moderate and high PAL group(P<0.01). The means of NMD (%) is 25.13%±6.52%,24.38%±5.44% and 29.50%±7.25% respectively (P> 0.05) and there was no significant difference among three groups. There was no positive relation between PAL and FMD in premenopausal females but in men and postmenopausal females. Although individuals of high PAL have the best FMD, the moderate PAL can also retard FMD decrease with ageing. Conclusion: Maintaining high physical activity level can enhance endothelial-dependent vascular dilation, and moderate or high physical activity level can prevent endothelial-dependent vaseular dilation declining with aging, which may contribute to decrease risk of cardiovascular disease in healthy middle-aged adults.