Our previous study has demonstrated aerobic exercise contributed to the improvement of endothelial vasodilation functions by nitric oxide (NO) signaling activation during pre-diabetes mellitus. The present study was designed to examine the contribution of hypoxia inducible factor (HIF)-1alpha to the regulation of endothelial nitric oxide synthase (eNOS) expressions in the vascular endothelium of prediabetic rats with HIF-1alpha specific inhibitor echinomycin (Ech) and/or aerobic exercise. The results showed the expressions of eNOS mRNA and protein dramatically decreased after Ech treatment (p<0.05), while significantly increased exercise (p<0.05). Interestingly, no obvious changes of HIF-1alpha mRNA and protein expressions were found after Ech treatment. Further investigation found HIF-1 binding activity was inhibited after Ech treatment, which may contribute to the decrease of eNOS expressions. Together, the present study clearly indicated aerobic exercise induced eNOS expressions through the activation of HIF-1alpha signaling in the vascular endothelium of rat aorta during prediabetes mellitus, which shed a light on further understanding the protective effects of long-term habitual physical activity against vasodilation dysfunctions during pre-diabetes mellitus.