Acute mountain sickness is thought to be triggered by cerebral hypoxemia and be prevented by acetazolamide (Actz). The effect of Actz on cerebral oxygenation at altitude remains unknown. In 16 members of the 2005 Dutch Cho Oyu (8201 m, Tibet) expedition, the influence of Actz and exercise (750 mg PO daily) on heart rate, peripheral and regional cerebral oxygen saturation (SaO2 and rSO2, the Lake Louise score (LLS), and psychomotor function were studied at 0 m 14 days prior to the expedition, after arrival at 3700 m on day 3, after arrival at 5700 m on day 29, and again at 5700 m before the end of the expedition on day 51. After arrival at 3700 m, the LLS of the climbers taking Actz (n = 8) was significantly lower compared to those who did not take Actz (n = 8): 0.75 ± 1.0 versus 2.9 ± 2.0, p < 0.05 (ANOVA). High LLSs were associated with low rSO2 values in rest and exercise (p < 0.01 and p < 0.001). With altitude, resting SaO2 and resting rSO2 decreased significantly (p < 0.001), irrespective of Actz use. Exercise at 3700 m and 5700 m reduced SaO2 and rSO2 even further compared to rest (p < 0.001), although at 3700 m the rSO2 was preserved better in those who took Actz (55.3 ± 4.3% versus 47.9 ± 5.7%, p < 0.05). Irrespective of Actz use, with altitude, the percentage of omissions in the vigilance and tracking test increased while the climbers' scores on vigor decreased (p < 0.05). In conclusion, at altitude, exercise-induced reduction in cerebral oxygenation is less in climbers on Actz compared to climbers not taking Actz. This effect is nullified after several weeks at altitude due to acclimatization in climbers not taking Actz.