Background: In major depressive disorder (MDD), findings include hyperstable regulation of brain arousal measured by electroencephalography (EEG) vigilance analysis and alterations in serum levels of cytokines. It is also known that cytokines affect sleep-wake regulation. This study investigated the relationship between cytokines and EEG vigilance in participants with MDD and nondepressed controls, and the influence of cytokines on differences in vigilance between the two groups. Methods: In 60 patients with MDD and 129 controls, 15-min resting-state EEG recordings were performed and vigilance was automatically assessed with the VIGALL 2.0 (Vigilance Algorithm Leipzig). Serum levels of the wakefulness-promoting cytokines interleukin (IL)-4, IL-10, IL-13 and somnogenic cytokines tumor necrosis factor-α, interferon-γ and IL-2 were measured prior to the EEG. Results: Summed wakefulness-promoting cytokines, but not somnogenic cytokines, were significantly associated with the time course of EEG vigilance in the MDD group only. In both groups, IL-13 was significantly associated with the course of EEG vigilance. In MDD compared to controls, a hyperstable EEG vigilance regulation was found, significant for group and group × time course interaction. After controlling for wakefulness-promoting cytokines, differences in vigilance regulation between groups remained significant. Conclusions: The present study demonstrated a relationship between wakefulness-promoting cytokines and objectively measured EEG vigilance as an indicator for brain arousal. Altered brain arousal regulation in MDD gives support for future evaluation of vigilance measures as a biomarker in MDD. Since interactions between cytokines and EEG vigilance only moderately differed between the groups and cytokine levels could not explain the group differences in EEG vigilance regulation, cytokines and brain arousal regulation are likely to be associated with MDD in independent ways.