The protective effects of exercise training on the diabetic-induced endothelial cell (EC) dysfunction were determined using intravital fluorescent microscopy. Male Sprague-Dawley rats were divided into three groups of control (Con), diabetes (DM), and diabetes with exercise--training (DM+Ex). Diabetes was induced by single intravenous injection of streptozotocin (STZ; 50 mg/kg BW). The exercise training protocol consisted of treadmill running, 5 times/week with the velocity of 13-15 m/min, 30 min/day periods for 12 and 24 weeks (wks). 24 wks after the STZ injection, blood glucose (BG), glycosylated hemoglobin (HbA1C), mean arterial blood pressure (MAP) and heart weight (HW) were significantly higher in DM rats (p < 0.001). However, DM+Ex rats had reduced the abnormalities of MAP (p < 0.01) and HW (p < 0.05) compared with DM rats. Furthermore, there was a significant decrease in heart rate (HR) of DM+Ex rats (p < 0.05) relative to Con rats. To examine the influence of exercise training on EC dysfunction, leukocyte-EC interactions in mesenteric venules and vascular reactivity responses to vasodilators in mesenteric arterioles were monitored by using intravital fluorescence microscopy. The diabetic state enhanced leukocyte adhesion in mesenteric postcapillary venules (p < 0.001). Moreover, an impaired vasodilatory response to the EC-dependent vasodilator, acetylcholine (Ach), not to sodium nitroprusside (SNP), was found in 12- and 24-wk diabetic rats (p < 0.01). The leukocyte adhesion and the impairment of EC-dependent vasodilation to Ach were attenuated by exercise training (p < 0.05). In addition, exercise training was also shown to have favorable preventive effects on hyperglycemia induced oxidative stress, as lower malondialdehyde (MDA) levels were observed from both groups of 12 and 24 weeks DM+Ex compared with DM (p < 0.01). In conclusion, our findings indicate that the endothelial dysfunction of diabetic rats could be characterized by increased leukocyte adhesion and impaired endothelium-dependent relaxation. Regular low intensity exercise training could improve both indices of endothelial dysfunction through amelioration of diabetic-induced oxidant/antioxidant levels. These findings support the notion that regular exercise training could be a fundamental form of therapy in preventing diabetic cardiovascular complications potentiated by endothelial dysfunction.