(1) Cerebral arteriolar vasomotor function is vital for brain health and has been examined through CO2 inhalation or breath-holding, which are both challenging for patients. We have developed a non-invasive method to evaluate this function with magnetic resonance imaging (MRI) by utilizing respiration-induced natural changes in partial pressure of arterial CO2 (PaCO2). In this study, we applied this method for 20s to evaluate the chronic effect of a few years smoking on the cerebral arteriolar vasomotor function. (2) A single slice (five slice thicknesses: 15 mm to 7 mm) perpendicular to the superior sagittal sinus of was imaged successively for 45 s using spin-echo echo-planar imaging by 3T MRI for ten smokers (24.5 ± 1.6 years) and ten non-smokers (24.3 ± 1.4 years), respectively. The venous oxygenation fluctuation (ΔYr) caused by the respiration-induced changes of PaCO2, which reflects the arteriolar vasomotor function, was calculated from the time series MR signal changes of superior sagittal sinus. (3) The ΔYr values of the smokers (0.7 ± 0.6) were significantly lower than those of the non-smokers (1.3 ± 0.8) (p = 0.04). (4) Degeneration of the cerebral arteriolar vasomotor function due to chronic smoking (even after 20s) was demonstrated by our non-invasive MRI-based method.