We aimed to investigate the effects of a single carbon dioxide (CO₂) treatment on arterial stiffness by monitoring the changes of aortic pulse-wave velocity (PWV) and aortic augmentation index (AIXao), which are indicators of arterial stiffness. PWV and AIXao were measured by an invasively validated oscillometric device. The measurements of stiffness parameters were performed before the CO₂ treatment, and at 1, 4 and 8 h after the first treatment. Thirty-one patients were included. No significant changes were found in PWV. AIXao decreased significantly 1 h and 4 h after CO₂ treatment compared to baseline values (p=0.034 and p<0.001). AIXao increased 8 h after the CO₂ treatment, but remained significantly lower than baseline AIXao values (p=0.016). CO₂ treatment is capable of reducing peripheral vascular resistance. We hypothesize that CO₂ is not only a temporal vasodilator but is also capable of activating vasodilation pathways.