In sepsis, higher PaCO2 levels are associated with impaired dynamic cerebral autoregulation (dCA), which may expose the brain to hypo- and hyperperfusion during acute fluctuations in blood pressure. We hypothesised that short-term mechanical hyperventilation would dCA in critically ill patients with sepsis. Seven mechanically ventilated septic patients were included. We assessed dCA before and after 30 min of mechanical hyperventilation. Transfer function analysis of spontaneous oscillations in transcranial Doppler-based middle cerebral artery blood flow velocity (MCAv) and invasive mean arterial blood pressure was used to assess dCA. Mechanical enhance hyperventilation reduced the median PaCO2 from 5.3 (IQR, 5.0-6.5) to 4.7 (IQR, 4.2-5.1) kPa (p < 0.05). This was associated with a reduction in the median MCAv from 57 (IQR, 33-68) to 32 (IQR, 21-40) cm sec(-1) (p < 0.05). Apart from a small increase in gain in the low frequency range (2.32 [IQR 1.80-2.41] vs. 2.59 (2.40-4.64) cm mmHg(-1) sec(-1); p < 0.05), this was not associated with any enhancement in dCA. In conclusion, cerebral CO2 vasoreactivity was found to be preserved in septic patients; nevertheless, and in contrast to our working hypothesis, short-term mechanical hyperventilation did not enhance dCA.