Previous studies have demonstrated that the activation of orexinergic neurons facilitates the recovery of animals from general anesthesia. Moreover, serotonergic neurons that receive projections from orexin neurons have also been shown to participate in sleep-wakefulness regulation. In the present study, we aimed to explore whether orexinergic neurons facilitate emergence from isoflurane anesthesia in rats by activating serotonergic neurons. Orexin A (30 or 100 pmol), orexin B (30 or 100 pmol), and their respective antagonists SB-334867 and TCS-OX2-29 (5 or 20 μg) were microinjected into the dorsal raphe nucleus (DRN) of rats, and their effects on induction and emergence times were analyzed. Electroencephalogram (EEG) changes were also recorded and analyzed to illuminate the effect of orexin injection into the DRN on cortical excitability under isoflurane anesthesia. Activation of serotonergic neurons was detected via immunohistochemical analysis of c-Fos expression following orexin administration. Our results indicated that injection of neither orexins nor orexin antagonists into the rat DRN exerted an impact on induction time, whereas orexin-A injection (100 pmol) enhanced arousal when compared with the saline group. In contrast, administration of orexin receptor type 1 antagonist SB-334867 (20 μg) prolonged emergence time from isoflurane anesthesia. Microinjection of orexin-A induced an arousal pattern on EEG, and decreased the burst suppression ratio under isoflurane anesthesia. Isoflurane anesthesia inhibited the activity of serotonergic neurons, as shown by decrease in the number of c-Fos-immunoreactive serotonergic neurons when compared with the sham group. This inhibitory effect was partially reversed by administration of orexin-A. Taken together, our findings suggest that orexinergic signals facilitate emergence from isoflurane anesthesia, at least partially, by reversing the effects of isoflurane on serotonergic neurons of the DRN.