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General anesthesia is widely used in various clinical practices due to its ability to cause loss of consciousness. However, the exact mechanism of anesthesia-induced unconsciousness remains unclear.It is generally thought that arousal-related brain nuclei are involved. 5-hydroxytryptamine (5-HT) is closely associated with sleep arousal. Here, we explore the role of the 5-HT system in anesthetic awakening through pharmacological interventions and optogenetic techniques. Our data showed that exogenous administration of 5-hydroxytryptophan (5-HTP) and optogenetic activation of 5-HT neurons in the dorsal raphe nucleus (DR) could signi cantly shorten the emergence time of sevo urane anesthesia in mice, suggesting that regulation of the 5-HT system using both endogenous and exogenous approaches could mediate delayed emergence. In addition, we rst discovered that the different 5-HT receptors located in the DR, known as 5-HT autoreceptors, are essential for the regulation of general anesthetic awakening, with 5-HT1A and 5-HT2A/C receptors playing a regulatory role. These results can provide a reliable theoretical basis as well as potential targets for clinical intervention to prevent delayed emergence and some postoperative risks.New Century 131 outstanding young talent plan top-level of Hang Zhou to HHZ. Zhejiang Health Science and Technology Plan (Grant.NO:2022KY248) to XiaoLing Liu.