It has long been thought that the treatment of core anxiety symptoms will produce a satisfactory resolution of insomnia. Genistein, the principal isoflavone constituent of soybean, has been reported to exert anxiolytic-like effects in rats. Whether genistein modulates sleep behavior, however, remains to be elucidated. In the present study, we examined the effect of genistein on sleep–wake profiles in freely moving mice by EEG and EMG recording as well as immunohistochemical staining for c-Fos. Our results showed that genistein administered i.p. at doses of 80 and 160 mg/kg significantly increased the amount of non-rapid eye movement (non-REM, NREM) sleep and as well as the number of NREM sleep episodes, with a concomitant reduction of wakefulness. It increased the state transitions from wakefulness to NREM sleep and subsequently from NREM to wakefulness. However, genistein neither had an effect on the amount of REM sleep nor on EEG power density of NREM or REM sleep. Immunostaining revealed a reduction of c-Fos expression in neurons of the locus coeruleus and the tuberomammillary nucleus after administration of genistein, as compared with the vehicle control. These results indicate that treatment with genistein leads to an inhibition of arousal-promoting neurons and thus promotes NREM sleep, suggesting its potential application for the treatment of insomnia.