Background The present study aims to compare regional oxygen supply determined by Near-Infrared Spectroscopy in the course of pulsatile perfusion with non-pulsatile perfusion during cardiopulmonary bypass in patients undergoing valvular heart surgery. Methods In this prospective randomized single-blinded trial, we enrolled adult subjects aged 18–65 years scheduled for elective valvular heart repair/replacement surgery with non-stenotic carotid arteries, employing a consecutive sampling method. Eligible patients were then randomly assigned in a 1:1 ratio to pulsatile or non-pulsatile perfusion during aortic cross-clamp. The primary outcome was regional cerebral oxygenation monitored by Near-Infrared Spectroscopy in each group. Results Seventy patients were randomly assigned, and each group comprised 35 patients. Mean age was 46.8 and 46.5 years in pulsatile and non-pulsatile groups, respectively. There were no significant between-group differences in regional cerebral oxygen saturation at different time points of cardiopulmonary bypass ( p-value for analysis of variance repeated measures: 0.923 and 0.223 for left and right hemispheres, respectively). Moreover, no significant differences in regional cerebral oxygen saturation levels from baseline between pulsatile and non-pulsatile groups at all desired time points for the left ( p = 0.51) and right ( p = 0.22) hemispheres of the brain were detected. Conclusion Pulsatile perfusion during cardiopulmonary bypass does not offer superior regional cerebral oxygenation measured by Near-Infrared Spectroscopy than non-pulsatile perfusion during cardiopulmonary bypass. Nonetheless, the efficacy of pulsatile flow in the subgroup of patients in whom cerebral blood flow is impaired due to carotid artery stenosis needs to be explored and evaluated by this method in future studies.