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(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 (Δ<i>Y_r</i>) 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 Δ<i>Y_r</i> values of the smokers (0.7 ± 0.6) were significantly lower than those of the non-smokers (1.3 ± 0.8) (<i>p</i> = 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.