AbstractThe brain’s arousal state is controlled by several neuromodulatory nuclei known to substantially influence cognition and mental well-being. Here we investigate whether human participants can gain volitional control of their arousal state using a pupil-based biofeedback approach. Our approach inverts a mechanism suggested by previous literature that links activity of the locus coeruleus, one of the key regulators of central arousal and pupil dynamics. We show that pupil-based biofeedback enables participants to acquire volitional control of pupil size. Applying pupil self-regulation systematically modulates activity of the locus coeruleus and other brainstem structures involved in arousal control. Furthermore, it modulates cardiovascular measures such as heart rate, and behavioural and psychophysiological responses during an oddball task. We provide evidence that pupil-based biofeedback makes the brain’s arousal system accessible to volitional control, a finding that has tremendous potential for translation to behavioural and clinical applications across various domains, including stress-related and anxiety disorders.