Objective To investigate the role of glutamatergic neuron of paraventricular nucleus of the thalamus (PVT) in regulating s‑ketamine induced anesthesia. Methods In the current study, male Vglut2‑cre transgenic mice, aged 8 to 10 weeks, were selected. Three of the mice were stereotaxically injected with calcium signaling virus rAAV‐EF1α‐DIO‐GCaMp6s‐WPRE‐hGH pA in PVT area, after 3 weeks calcium imaging and fiber photometry were used to record the activity of PVT glutamatergic neurons before and after anesthesia with s‑ketamine. According to the random number table method, ten mice were divided into two groups (n=5): a ChR2 group and a mCherry1 group. Excitatory optogenetic virus rAAV‐EF1α‐DIO‐hChR2（H134R）‐mCherry‐WPRE‐hGH pA or control virus rAAV‐EF1a‐mCherry‐WPRE‐hGH pA was stereotaxically injected into PVT area, after 3 weeks optogenetics was used to activate PVT glutamatergic neurons. The changes of EEG spectrum and percentage of total power of each frequency band in the mCherry1 group and ChR2 group were observed. Furthermore, according to the random number table method, 16 mice were divided into two groups (n=8): a chemogenetic inhibition (hM4Di) group and a control virus (mCherry2) group. Inhibitory chemogenetic virus rAAV‐EF1α‐DIO‐hM4D（Gi）‐mCherry‐WPREs pA or control virus was stereotaxically injected into PVT area after 3 weeks. The glutamatergic neurons of PVT were inhibited by chemogenetic technique, and the induction and emergence time in the mCherry2 group and hM4Di group were observed. Results Compared with the awake baseline, the calcium signal of glutamatergic neurons in PVT increased significantly during induction period of s‑ketamine anesthesia (P<0.05), lasting about 300‒500 s. During anesthesia state, the calcium signal decreased significantly (P<0.05). There was no statistical significance in calcium signal after recovery of righting reflex (RORR) compared with anesthesia state (P>0.05), but it was lower than awake baseline (P<0.05). PVT glutamatergic neurons were activated by optogenetics technique, and the percentage of total power in δ band in ChR2 group was significantly decreased compared with that before stimulation (P<0.05), the percentage of total power in α band increased significantly (P<0.05), and there was no significant difference in other frequency bands (P>0.05). Chemogenetic inhibition of PVT glutamate neurons, compared with mCherry2 group, hM4Di group had longer emergence time (P<0.01), but there was no significant difference in induction time (P>0.05). Conclusions PVT glutamatergic neurons are involved in the anesthesia and arousal process of s‑ketamine. Activation of PVT glutamatergic neurons can promote emergence from anesthesia induced by s‑ketamine.